Transgenic plants expressing CIVPS or intein modified proteins and related method

ABSTRACT

Transgenic plants that express CIVPS or intein modified proteins, compositions of matter comprising them, products of diverse applications made from the transgenic plants, methods to construct the transgenic plants containing CIVPS or intein modified genes, methods to express CIVPS or intein modified proteins in plants, and methods of using the transgenic plants.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US03/00432 filed Jan. 7, 2003 which in turn claims priority fromU.S. provisional application No. 60/346,541 filed Jan. 8, 2002, whichare both incorporated by reference as if fully set forth.

FIELD OF THE INVENTION

The present invention relates to transgenic plants expressing CIVPS orintein modified proteins, methods for the production of the transgenicplants, methods for the expression of CIVPS or intein modified proteinsin plants, and various uses of and products containing the transgenicplants expressing CIVPS or intein modified proteins.

BACKGROUND

Since fossil fuels are non-renewable resources, adequate supplies ofenergy and organic feedstocks need to be secured for the future. Atransition to sustainable resources requires new technologies for theconstruction of improved feedstocks, the design of efficient processesto convert the feedstocks into valuable products, and/or the design ofproducts that efficiently utilize an altered substrate spectrum. Thistransformation will create benefits such as decreased pollution fromenergy production and use, decreased pollution from chemicalmanufacturing processes, increased sustainability through theutilization of renewable natural resources and organic waste products assubstrates, decreased dependence on foreign country's raw materials, andan increase in local economies and markets involved in the production ofnew substrates.

Plant biomass is one sustainable resource that can help meet futurefeedstock requirements. The use of plants as substrates for energy,chemical, pharmaceutical, and organic feedstock takes advantage ofexisting large-scale agricultural production, uses energy from the sunto incorporate carbon dioxide into plants via photosynthesis, and hasfewer environmentally hazardous by-products. By using photosynthesis,plants make the carbon dioxide removed from the air available for theproduction of energy, chemicals, and agricultural products. Finding waysto effectively redistribute this carbon in forms that are readily andeconomically employable remains a challenge.

The production of chemical feedstocks and fuels from plant biomass isstill in its infancy. Starch-based raw materials, for example, may beapplied to the production of commodity or specialty chemical products.Poor substrate and strain availability hampering bioconversion, alongwith real or perceived safety issues related to containment, and a lackof economic viability, have made progress in this area particularlyslow. Non-cellulosic biomass, such as corn starch, compares favorablywith fossil resources on a mass basis, but is too costly. Cellulosicbiomass, such as short-rotation poplar, pine, switchgrass, corn stover,sugar cane bagasse, waste paper sludge, and municipal solid waste, incontrast, is cost competitive in terms of both mass and energy.Cellulosic biomass, because of its complex structure, is neverthelessdifficult to process. Currently, cellulosic biomass requirespretreatment with strong acids, bases, and/or other chemicals for use asa substrate for fuel, e.g. ethanol, or for chemical production, e.g.paper products. This pretreatment efficiently exposes polymericsubunits, primarily hexoses, pentoses, and phenolic compounds, which arethen cleaved and used as substrates, but is expensive. One alternativeto the use of more hazardous chemicals is the use of enzymes, althoughit is less cost effective.

Recombinant DNA technology has been applied to alter microorganisms toperform substrate bioconversion at reduced costs, thus expanding the useof microorganisms, and increasing the number of products that areproduced. For example, plant cells that express lignocellulosicdegrading enzymes have been constructed, although they rarelydifferentiate and regenerate into complete plants due to decompositionof structural components. In cases where they differentiate intocomplete plants, e.g. with lignin and cellulose substrates, the enzymeactivities are low and the plants require further processing. Attemptsto combine pretreatment of substrate biomass with fermentation haveencountered difficulties as well, in part because of mass transferlimitations and interference with the fermenting organism.

CIVPS or inteins are in-frame, self-cleaving peptides that generallyoccur as part of a larger precursor protein molecule. CIVPS or inteinsdiffer from other proteases or zymogens in several fundamental ways.Unlike proteases that cleave themselves or other proteins into multiple,unligated polypeptides, CIVPS or inteins have the ability to both cleaveand ligate in either cis or trans conformations. Thus as opposed toterminal cleavage that would result from the reaction of a protease on aprotein, CIVPS or inteins have the ability to cleave at multiple sites,and ligate the resulting protein fragments. This cleavage is inducedunder specific conditions and can be engineered using molecular biologytechniques. CIVPS or inteins have been described in the literature inSacchromyces cerevisiae (Kane et. al., Science 250:651; Hirata et al.,J. Bio. Chem. 265:6726 (1990)), Mycobacterium tuberculosis (Davis etal., J. Bact. 173:5653 (1991), Davis et al., Cell 71:1 (1992)),Thermococcus litoralis (Perler, et al., PNAS 89:5577 (1992)), and inother organisms, but do not occur naturally in plants.

Accordingly, there is a need for providing novel methods for producingenergy and other pharmaceutical or industrial products from more easilyrenewable sources, such as by modifying plants in a manner such thatthey may be used as energy and chemical feedstocks.

SUMMARY

The present invention provides for genetically recombinant plants, theirparts, plantlets, seeds, seedlings, and their progeny (collectivelyreferred to as “plants”), which may contain single or multiple exogenousgene sequences, each being interrupted by, or fused to single ormultiple Controllable InterVening Protein Sequence (CIVPS) or inteinsequences, or a combination of a CIVPS or intein sequence, andoptionally regulatory sequences suitable for gene expression andtransformation of a plant. The modified gene sequences may be expressedconstitutively or transiently, throughout the entire plant or inspecific tissues, or any combination thereof encompassing both singleand multiple CIVPS or intein modified gene sequences. In differentembodiments of the invention, any modified gene sequence, or set ofmodified gene sequences, may be expressed in any or all tissuesconstitutively or at specific times.

The invention also relates to methods of producing transgenic plantscomprising CIVPS or intein modified genes, e. g. by first constructing apiece of DNA comprising the parent CIVPS or intein modified gene, andtransforming the plant with the construct.

The invention also relates to methods of producing an CIVPS or inteinmodified protein(s) in transgenic plants, e. g. by transforming theplant, or plant cells, with a single or multiple modified genesequence(s), and expressing the CIVPS or intein modified protein(s). Inone preferred embodiment the gene sequences may be expressed at anytime. In another embodiment, prior to the protein(s) being spliced itpreferably is(are) provided with a substantially different activity(ies)and/or structural property(ies). The spliced protein product(s)has(have) its(their) activity(ies) unveiled, unless inhibited by anexogeneously added or endogeneously produced molecule(s) analogous tothe non-CIVPS or intein modified protein parent sequence. The CIVPS orintein modified gene products may be expressed in large quantities andrecovered from the plant material. Alternatively, the plant or plantmaterial may itself be used as a source of CIVPS or intein modified geneproducts.

The invention also provides for the use of CIVPS or intein modified geneproducts expressed in plants, the use of transgenic plants expressingCIVPS or intein modified genes in animal feed, or the use of transgenicplants expressing CIVPS or intein modified genes in batch, semi-batch,and continuous industrial processes for the production of fuels,chemical products, animal food or food additives, pharmaceuticals,paper, paper products, and for vaccine delivery and the remediation ofwaste materials.

Other objects, advantages and features of the present invention willbecome apparent to those skilled in the art from the following briefdescription of the drawings and discussion.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIG. 1 illustrates the construction of an CIVPS or intein modifiedprotein coding DNA sequence by constructing an CIVPS or intein modifiedprotein DNA coding sequence constructed by fusion of an CIVPS or inteincoding sequence to the coding sequence of a protein of a purportedactivity, at either the 3′ end of the gene, the 5′ end of the gene, orinternally, within the protein gene. Other variants are possible bycombining any of the three resulting CIVPS or intein modified proteincoding sequences shown in FIG. 1.

FIG. 2 illustrates one configuration of the resulting CIVPS or inteinmodified proteins, or components thereof. This figure demonstrates thecase of a single CIVPS or intein modified protein. Multiple nativeprotein sequences, however, may be combined with single or multipleCIVPS or inteins as well.

FIG. 3 illustrates the cleavage of an CIVPS or intein modified protein,or components thereof, which may be attained in vitro or in vivo whensubjected to an appropriate cleavage stimulus(i). Illustrated hereschematically is an example of the cleavage process for a single CIVPSor intein modified protein. Other variants may be constructed ascombinations of the CIVPS or intein modified proteins shown in thisfigure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

This invention arose from a desire by the inventor to provide novelmethods for generating valuable products from renewable resources, e. g.plant materials or biomass, and to do this in a cost effective manner.One way to effectively attain this goal is by modifying plant biomassthrough the use of CIVPS or intein modified proteins, where the CIVPS orintein is attached to a desired protein. Within the text of this patentthe terms CIVPS and intein are intended to refer to similar products,and will be used interchangeably. From the knowledge that inteinmodified proteins may be expressed in cells at high titer, yet withsubstantially decreased activity, he concluded that, if cloned into aplant, this decrease in activity would allow the thus formed transgenicplant cells, plant fragments, or plant tissues, to develop into inteinmodified protein producing complete plants. Moreover, he thought thatsuch transgenic plants could be provided as several differentembodiments, such as those where the recombinant plants are made toexpress the modified proteins either 1) constitutively or transiently,2) through chemical induction or biological induction by the plant'sgrowth cycle, 3) throughout the entire plant or specifically in distinctplant tissues, and/or 4) with or without subcellular localization, amongothers. As envisioned by the inventor, in one embodiment of thisinvention, the expressed intein modified protein(s) is(are) comprised ofa parent protein sequence(s), whose activity(ies) may be known, inferredthrough sequence or structure homology and/or produced by mutagenesis orby de novo synthesis; each parent sequence(s) being interrupted by, orfused to, an intein sequence(s) or portions thereof. Once inserted, theintein portion(s) of the modified protein(s) inactivate(s), in vivo, theactivity or structural utility of the parent protein. The parentprotein's original activity may be, however, substantially recovered, ifand when desired, by induction of intein splicing. For example, in oneapplication, following plant harvest and during substrate pretreatment,each CIVPS may be induced to splice itself from its parent proteinsequence, which parent protein now has recovered its original activity.Methods for intein splicing with, or without, recombining of the proteinto a functioning activity are known to one skilled in the art, and neednot be repeated here. These methods include the use of light,temperature, change in pH, and/or the addition of chemical reagents.

More specifically, this invention is directed to a recombinant plant, orplant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent, comprising an expressionconstruct(s) that encode(s) at least one modified protein comprising atarget protein(s) or protein segment(s), which is(are) fused, eitherinternally or terminally, to a controllable intervening proteinsequence(s) (CIVPS) or intein sequence(s) or segment(s) thereof, or toan amino terminus(i) or a carboxyl terminus(i) thereof. In oneembodiment, each expression construct of the plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent comprises, operatively linked to oneanother, a first nucleic acid segment(s) encoding a target protein(s),and a second nucleic acid segment(s) encoding a CIVPS or inteinsequence(s), and optionally a selectable marker(s) or reporter gene(s)and/or a promoter(s). It is understood that in a more specificembodiment the sequences may be fused, either directly or via alinker(s), and more preferably in reading frame. The modified protein(s)may be expressed by the plant, or plant part, plantlet, tissue, cell,sub-cellular fraction, seed, seedling, protoplast, progeny or descendenteither constitutively, or inductively. In the latter case, theexpression and/or splicing of the at least one modified protein(s) maybe triggered or induced by a stimulus(i). Examples of suitable stimulicomprise a pH change, change in osmolality, or temperature, the additionof a fertilizer, pesticide, or chemical, or a change in light, and/orsound. The plant, or plant part, plantlet, tissue, cell, sub-cellularfraction, seed, seedling, protoplast, progeny or descendent may expressthe modified protein(s) either at a pre-determined point of the plantlife cycle, in one or more specific tissues or parts thereof, and/or inat least one specific sub-cellular compartment(s). Alternatively or inconjunction with the latter the modified protein(s) may be expressed andsecreted extracellularly. The plant, or plant part, plantlet, tissue,cell, sub-cellular fraction, seed, seedling, protoplast, progeny ordescendent specific tissue(s) may be seeds, roots, fruits, stems, tubersand/or leaves, and the specific subcellular compartments may be acellular apoplast, cytosol, chloroplast, plastid, endoplasmic reticulum,inclusion body, vacuole and/or nucleus. Other variations, however, arealso included within the confines of this invention.

The plant, or plant part, plantlet, tissue, cell, sub-cellular fraction,seed, seedling, protoplast, progeny or descendent may also carry aselectable marker that confers it resistance to a chemical. Examples ofthese are bromoxynil, 2,2-dichloropropionic acid, G418, glyphosphate,haloxyfop, hygromycin, imidazoline, kanamycin, methotrexate, neomycin,phosphinothricin, sethoxydim, 2,2-dichloropropionic acid, glyphosphate,hygromycin, trichothecne, sulfonylurea, s-triazine, and/ortriazolopyrimidine. Others, however, may also be employed. The promotermay be included to precede a CIVPS or intein-modified proteinpolynucleotide. In some cases, the plant, or plant part, plantlet,tissue, cell, sub-cellular fraction, seed, seedling, protoplast, progenyor descendent may be tolerant or resistant to normally extremely toxiclevels of a selected chemical(s). In another embodiment, the plant, orplant part, plantlet, tissue, cell, subcellular fraction, seed,seedling, protoplast, progeny or descendent is fertile, and has at leastone heritable modified protein encoding polynucleotide sequence(s).However, it may just as well not be fertile. Further, as indicatedabove, also part of this invention are inbred and hybrid geneticallyrecombinant plants, or plant parts, plantlets, tissues, cells,sub-cellular fractions, seeds, seedlings, protoplasts, progeny anddescendents, which may or may not be produced by the method of thisinvention. Of particular interest are plant parts, plant seeds, plantseedlings and plant protoplasts, which have substantial commercialimportance. Also of commercial and other interest are plants, planttissues, plant cells, and sub-cellular fractions. The spliced proteinmay have the ability of changing the content or activity of one or moreplant component(s). In one example, the content may be altered, e.g.reduced, of a plant component such as glucose, fructose, cellulose,hemicellulose, lignin, glycerol, glycine-betaine, pectin, sucrose,lactose, maltose, galactose, amino acids, lipids, vitamins and/orstarch, and the like. In another, the plant component whose activity isaltered, e.g. reduced, may be one or more of proteins, RNA, and/orlipids, among others. In one aspect, the CIVPS or intein sequence andthe target protein or protein segment form at least one splice junctionwith the target protein. In a desirable embodiment, the amino acidresidue at the carboxyl terminus(i) of the splice junction(s) is(are)provided with a hydroxyl or a sulfhydryl side chain(s). In anotherparticularly useful embodiment, the splice junction(s) is placeddownstream of the CIVPS or intein sequence(s) or segment(s) thereof, andmay comprise(s) an amino acid residue(s) lacking, for example, hydroxylor sulfhydryl side chains at the amino terminus(i) of the target proteinor protein segment(s). In another important variation, the splicejunction(s) is(are) placed upstream of the CIVPS or intein sequence(s)or segment(s) thereof, and may comprise an amino acid residue(s) havinghydroxyl or sulfhydryl side chains at the amino terminus(i) of the CIVPSor intein sequence(s) or segment(s) thereof. Another importantpossibility is that where the splice junction(s) is(are) placed upstreamof the CIVPS or intein sequence(s) or segment(s) thereof, and it maycomprise(s) a cysteine. Still another important variation is thatwherein the splice junction(s) is(are) placed downstream of the CIVPS orintein sequence(s) or segment(s) thereof, and may be provided withHis-Asn at the carboxyl terminus(i) of the CIVPS or intein sequence(s)or segment(s) thereof, and/or with an amino acid residue(s) havinghydroxyl or sulfhydryl side chains at the amino terminus(i) of theadjoining region(s) of the target protein(s). In yet another interestingvariant, the splice junction(s) is placed downstream of the CIVPS orintein sequence(s) or protein segment(s) thereof, and may be providedwith an Asp at the carboxyl terminus(i) of the CIVPS or inteinsequence(s) or segment(s) thereof, and/or with an amino acid residue(s)having hydroxyl or sulfhydryl side chains at the amino terminus(i) ofthe adjoining region(s) of the target protein(s) or protein segment(s).Further modifications are those where the Asp at the carboxylterminus(i) is replaced by an amino acid(s) lacking carboxyl or aminoside chains, and where the CIVPS or intein sequence(s) or its segment(s)comprise(s) an externally controllable CIVPS or intein sequence(s) orsegment(s) thereof, which may be from, among other species, aSaccharomyces fungi, and more specifically a Saccharomyces cerevisiaefungi. Other constructs suitable for insertion in the products of theinvention are those where the CIVPS or intein sequence(s) or segment(s)thereof is(are) inserted immediately before Ser, Thr or Cys of thetarget protein(s) or protein segment(s), and where the CIVPS or inteinamino or carboxy terminus(s) comprise(s) Ser, Thr or Cys, among others.As described in more detail below, the target protein may be expressedin a microorganism, such as a bacterium, as is known in the art.Examples of microorganisms that may be employed are Bacillusthuringiensis, or Phytolacca insularis. One preferred target protein isBacillus thuringensis endotoxin, which results in a modified Bacillusthuringiensis endotoxin being expressed. Another embodiment includes theexpression of a target protein from a virus. Although any virus could beemployed, examples are potato virus Y, geminivirus, aspermy virus 2b,and cucumber mosaic virus, among others. Another embodiment includes theexpression of human target proteins. Although any human protein could beused, examples of preferred proteins include insulin, erythropoietin,growth hormone, tumor necrosis factor receptor, leptin, and otherproteins of therapeutic value.

The recombinant plant, or plant part, plantlet, tissue, cell,sub-cellular fraction, seed, seedling, protoplast, progeny or descendentmay be produced by a method comprising

-   -   providing an expression construct that encode(s) at least one        modified protein comprising a target protein, or protein        segment(s), which is(are) fused, either internally or        terminally, to a CIVPS or intein sequence(s) or segment(s)        thereof, or to an amino terminus(i) or a carboxyl terminus(i)        thereof;    -   transforming a plant, or plant part, plantlet, tissue, cell,        sub-cellular fraction, seed, seedling, protoplast, progeny or        descendent, with the expression construct; and    -   regenerating a genetically recombinant plant, or plant part,        plantlet, tissue, cell, sub-cellular fraction, seed, seedling,        protoplast, progeny or descendent, from the transformed plant,        or plant part, plantlet, tissue, cell, sub-cellular fraction,        seed, seedling, protoplast, progeny or descendent, that        encode(s) at least one modified protein sequence(s).

It is highly preferred that the transformation be a stabletransformation. However, transformations that have some temporarystability are also desirable. The regeneration step may be conducted bybreeding of a recombinant plant, or plant part, plantlet, tissue, cell,sub-cellular fraction, seed, seedling protoplast, progeny or descendent;crossing of a recombinant plant, or plant part, plantlet, tissue, cell,sub-cellular fraction, seed, seedling, protoplast, progeny or descendentand a non-genetically recombinant plant, or plant part, plantlet,tissue, cell, sub-cellular fraction, seed, seedling, protoplast, progenyor descendent; and/or back-crossing of two genetically recombinantplant, or plant part, plantlet, tissue, cell, sub-cellular fraction,seed, seedling, protoplast, progeny or descendent. The expressionconstruct employed in this method may comprise one or more of promoter,selectable marker, resistance marker, heritable marker, poly-adenylationsequence, repressor, enhancer, localization sequence, and/or signalingsequence. These are intended for use in the application of recombinanttechnologies as is known in the art, and exemplified elsewhere and belowin the examples. In an important aspect of the method, the plant, orplant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent is(are) transformed with theexpression construct by either viral transformation, bombardment withDNA-coated microprojectiles, liposomal gene transformation, bacterialgene transfer, electroporation, or chemical gene transformation, or morethan one of these. As indicated above, the plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent, may be transformed by means of abacterium, e. g. Agrobacterium tumefaciens, although othermicroorganisms may also be employed. In the present method, thetransformation may be conducted by chemical gene transformation, and itmay be done with the aid of, e.g. calcium phosphate, and/or polyethyleneglycol, or other chemicals known in the art as being suitable for thispurpose. The selection may be attained with the aid of a selectablemarker, or a resistance marker, or of the expression of at least onenucleic acid encoding an CIVPS or intein modified protein. In the methodof the invention, the genetically recombinant plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent may be regenerated from a transformedembryogenic tissue(s); plant protoplasts; cells derived from immatureembryos; or from transformed seeds, among other sources.

Another method is also provided in this patent, which method is suitablefor producing a modified protein(s) or protein segment(s) from arecombinant transformed plant, or plant part, plantlet, tissue, cell,sub-cellular fraction, seed, seedling, protoplast, progeny or descendentexpressing the protein(s) or protein segment(s), that comprisesconducting the method described above, and further harvesting themodified protein(s) or protein segment(s) from the transformed plant, orplant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent. The method may furthercomprise purifying the modified protein, which may be done by one ofmany techniques known in the art. As described here, this method mayproduce a modified protein(s) or protein segment(s) that comprises aCIVPS or intein modified protein(s) or protein segment(s).

Still a further method is provided here for producing a modified proteincomprising a target protein(s) or protein segment(s) fused, eitherinternally or terminally, to a CIVPS or intein sequence(s0 or segment(s)thereof, or to its amino terminus(i) or carboxyl terminus(i), whichmethod comprises

-   -   obtaining an expression construct encoding a target protein        having an in-frame fused CIVPS or intein sequence(s) or        segment(s) thereof, or its amino terminus(i) or carboxyl        terminus(i);    -   transforming a host plant cell(s) with the expression construct;        and    -   culturing the transformed plant host cell under conditions        effective for expressing the modified protein.

In one preferred aspect, in the expression construct the at least onefirst nucleic acid segment(s) encoding the CIVPS or intein sequence(s)or segment(s) thereof is(are) fused to the 5′-end of the second nucleicacid segment(s) encoding the target protein(s) or protein segment(s).Alternatively, in the expression construct the first nucleic acidsegment(s) encoding the CIVPS or intein sequence(s) or segment(s)thereof may be fused to the 3′-end of the second nucleic acid segment(s)encoding the target protein(s) or protein segment(s). It is particularlysuitable to practice the present method to employ a Saccharomyces CIVPSor intein sequence(s) or segment(s) thereof, which is known to effect,either in cis or in trans, excision, cleavage, ligation,excision-ligation, cleavage-ligation, and/or cyclization. When the CIVPSor intein or its(their) segment(s)are employed to induce proteinsplicing, this event may be induced or triggered by a change oftemperature, light or pH, the addition/removal of a chemical reagentthat facilitates/inhibits splicing or cleavage, amino aciddephosphorylation or deglycosylation, or by contact with, or removal of,a peptide or peptidomimetic activating or blocking of splicing or ofcleavage. Another manner of inducing protein splicing is either in vitroor in vivo contact with, or removal of, a peptide or peptidomimeticagent that may either activate or block splicing or cleavage.Interesting variations that produce superior results are those where theamino or carboxy terminus(i) of the CIVPS or intein sequence(s) orsegment(s) thereof comprise(s) Ser, Thr or Cys, or where the carboxylterminus(i) of the CIVPS or intein sequence(s) or segment(s) thereofcomprise(s) Asp preceding Ser, Thr or Cys of the target protein(s) orprotein segment(s). However, other modifications are also possible, asis known in the art. See, for example, U.S. Pat. No. 5,834,247 thatdiscloses for the prokaryotic and eukaryotic realms some methodologyincorporated in this invention to the production of hybrid plants ofuseful characteristics. In the present method, the expression constructmay further comprise a promoter, a selectable marker, a resistancemarker, a heritable marker, a poly-adenylation sequence, a repressor, anenhancer, a localization sequence, or a signaling sequence. Moreover,the method presented here may also comprise the transformation of theplant, or plant part, plantlet, tissue, cell, sub-cellular fraction,seed, seedling, protoplast, progeny or descendent with the expressionconstruct being implemented by viral transformation, bombardment withDNA-coated microprojectiles, liposomal gene transfer, bacterial genetransfer, electroporation, and/or chemical gene transformation, and/orother methods known in the art, or that will be subsequently developed.As described above, in the method described here, the bacterium used totransfer the expression construct may be an Agrobacterium tumefaciensbacterium; the chemical used for transformation may be calciumphosphate, or polyethylene glycol; the transformed plant cells, plantparts, plants, etc. may be selected through their expression of aselectable marker, or resistance marker; the selection of thetransformed plant, or plant part, plantlet, tissue, cell, sub-cellularfraction, seed, seedling, protoplast, progeny or descendent may beconducted through their expression of the modified protein genesequence; and the regeneration of the genetically recombinant plant, orplant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent may be attained fromtransformed embryogenic tissue; from cells derived from immatureembryos; or from transformed seeds, among others.

Also disclosed in this patent is a method for producing seed thatexpress a modified protein(s), this method comprising

-   -   obtaining the genetically recombinant plant, or plant part,        plantlet, tissue, cell, sub-cellular fraction, seed, seedling,        protoplast, progeny or descendent of the invention;    -   culturing or cultivating the genetically recombinant plant, or        plant part, plantlet, tissue, cell, sub-cellular fraction, seed,        seedling, protoplast, progeny or descendent; and    -   obtaining from the cultivated plant seed that expresses a        modified protein(s).

Still another method provided by this patent is one for using a plant,or plant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent expressing a modifiedprotein for producing a compound, the method comprising

-   -   harvesting a recombinant plant, or plant part, plantlet, tissue,        cell, sub-cellular fraction, seed, seedling, protoplast, progeny        or descendent in accordance with the teachings of this patent;    -   mechanically processing the plant, or plant part, plantlet,        tissue, cell, sub-cellular fraction, seed, seedling, protoplast,        progeny or descendent;    -   combining the mechanically processed plant, or plant part,        plantlet, tissue, cell, sub-cellular fraction, seed, seedling,        protoplast, progeny or descendent, with a non-genetically        recombinant plant in a proportion greater than or equal to zero        recombinant:non-recombinant; and    -   chemically processing the plant or specific portions of the        plant under conditions effective for obtaining the compound.

This method may be practiced by mechanical processing of the plant, orplant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent by extrusion, grinding,shredding, mulching, chipping, dicing, compressing, exploding, and/ortearing. Other processing techniques, however, are also suitable. Thechemical processing of the combined components may be attained byvarious techniques or a combination thereof. Some of them arepre-treatment with steam, dilute or concentrated acid, ammoniaexplosion, sterilization, soaking in water, mixing with a solvent, achange of pH, temperature or osmolality, exposure to or changes inlight, inorganic and/or enzyme catalysis, saccharification, bleaching,scouring, fermentation, distillation, chromatography, adsorption, and/oraddition of a chemical(s). Others, of course, are also employedsuccessfully. Various steps are of use when practiced as follows: thepre-treatment may include steaming the combined products forsterilization purposes; the chemical processing may be attained bypre-treatment with at least one of sulfuric acid, hydrochloric acid,phosphoric acid, or carbonic acid, or by soaking in water at atemperature greater than or equal to about 20° C., and/or by mixing thecombined products with at least one of water, or an organic or inorganicsolvent(s). As already explained, an external stimulus(i) may be appliedto induce splicing of the modified protein(s) or protein segment(s).Examples of external stimuli are a change of pH, osmolality, ortemperature, exposure to sound, light, or addition of a chemical(s). Insome cases the spliced protein(s) or protein segment(s) may exhibitaltered activity(ies) with respect to the modified protein(s) or proteinsegment(s), such as altered binding, catabolic or anabolic activity(ies)with respect to the original target protein(s). Examples of splicedprotein(s) or protein segment(s)are those capable of degrading starch,dextrin, pectin, lipids, protein, chitin, lignin, cellulose, orhemicellulose, or modifying lignin, or having saccharification activity.Thus, the spliced protein may be capable of producing glucose, fructose,xylose, phenol, glycerol, mannose, lactic acid, acetic acid, ethylene,propylene, toluene, ethyl benzene, styrene, xylene, ethylene glycol,butadiene, formaldehyde, isopropanol, acetone, butanediol, methanol,ethanol, propanol, butanol, propanediol, vitamins, methane, ethane,propane, butane, pentane, hexane, heptane, octane, benzene, targetproteins, therapeutic proteins, enzymes and/or biopolymers, among othercompounds. In one specific embodiment of the pre-treatment,saccharification, and fermentation may be conducted in one step, and thefermentation may be attained by employing a prokaryotic or eukaryoticmicroorganism capable of producing lactic acid, acetic acid, ethylene,propylene, toluene, ethyl benzene, styrene, xylene, ethylene glycol,butadiene, formaldehyde, isopropanol, acetone, butanediol, methanol,ethanol, propanol, butanol, octanol, propanediol, vitamins, methane,ethane, propane, butane, pentane, hexane, heptane, octane, benzene,and/or biopolymers, among other compounds.

This invention also encompasses the production of animal feedstock thatcomprises a nutritious amount of the recombinant plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent of the invention. When the feedstockprovided by the inventor is ingested by an animal, the modifiedprotein(s) or protein segment(s) is(are) spliced by an internalstimulus(i) from the animal. Examples of internal stimuli are theanimal's saliva, bile, chymotrypsin, trypsin, bicarbonate, hydrochloricacid, or stomach pH or temperature, among others. The feedstock of theinvention may comprise spliced protein(s) such as phytases,endocellulases, exocellulases, amylases, glucanases, hemi-cellulases,pectinases, proteases, xylanases, or lipases, growth factors or a growthhormone. Other proteins, however, could also be employed as desired.

Yet another aspect of this invention provides for the use of thefeedstock described above in the manufacture of an immune responseenhancing composition, wherein the spliced protein(s) or proteinsegment(s) comprise(s) at least one recombinant immunogen(s). Theimmunogen may included one or more viral or bacterial immunogens, and itmay be formulated in various suitable forms. Preferred are an oralformulation, a trans-mucosal formulation, a gastrointestinal (G.I.)tract absorbed formulation. However, this composition of matter may beformulated in any systemic or topical form suitable for administrationto an animal, including its addition to animal feed.

The animal feedstock of the invention may be produced by firstconducting the steps indicated above to obtain a genetically recombinantplant, or plant part, plantlet, tissue, cell, sub-cellular fraction,seed, seedling, protoplast, progeny or descendent, and then processingthe genetically modified plant, or a portion of the resulting productunder conditions effective to obtain an animal digestible feedstock.

The product of this invention may also be employed for promoting animalgrowth, for example by producing feedstock that comprises a growthpromoting product, and allowing an animal access to the modifiedfeedstock. The product of this invention may also be employed forenhancing an animals immune response. This may be done by administeringto an animal in need of the treatment, an immune enhancing amount of thecomposition of the invention.

A further aspect of this invention involves a method for producing atarget protein(s) or protein segment(s), the method comprising

-   -   producing a first modified protein(s) or protein segment(s),        wherein the amino terminus of a CIVPS or intein sequence(s) or        segment(s) thereof is(are) fused to the carboxyl terminus(i) of        a target protein(s) or protein segment(s) by the method        described above;    -   producing a second modified protein(s) comprising a segment(s)        of the CIVPS or intein sequence(s); and    -   contacting first and second modified proteins under conditions        effective for trans cleavage of the CIVPS or intein sequence(s)        or segment(s) thereof by the second modified protein(s).

Yet another variation of the above method for producing a targetprotein(s), comprises

-   -   producing a first modified protein(s), wherein the carboxyl        terminus of a CIVPS or intein sequence(s) or protein segment(s)        thereof is(are) fused to the amino terminus(i) of the target        protein(s) or protein segment(s) by the already described        method;    -   similarly producing a second modified protein(s) or protein        segment(s) comprising a segment(s) of the CIVPS or intein        sequence(s); and    -   contacting first and second modified proteins under conditions        effective for trans cleaving the CIVPS or intein sequence(s) or        segment(s) thereof from the first modified protein(s) or protein        segment(s). The cleavage may be induced in this procedure by a        change in temperature, light, or pH, addition/removal of        chemical that facilitates/inhibits splicing or blocking of        cleavage, amino acid dephosphorylation or deglycosylation,        and/or contact/removal of peptide or peptidomimetic that        activates/blocks splicing/cleavage, among others.

Thus, the invention is directed towards transgenic plants, which term isintended in this patent to be synonymous with genetically recombinantplants, their seeds and progeny plants, or any plant portion, tissue orcell, containing a gene(s) for an CIVPS or intein modified protein(s).The invention is further directed towards methods for the production ofthe transgenic plants that produce CIVPS or intein modified proteins,methods for the production of CIVPS or intein modified proteins inplants, and uses of the plants as substrates for fuels, chemicals,animal food or food additives, paper, and pharmaceutical production. Theinvention allows for the production of transgenic plants that can beused as a source of binding, structural or catalytic components, or canhave their intein modified proteins purified and used separately asbinding, structural or catalytic proteins. Transgenic plants aremulti-cellular plants that express single or multiple exogenous genesand their associated protein (or ribonucleic acid) activities. Withinthe context of this invention, gene or enzyme classes may bespecifically referred to, however this is not a limiting aspect of theinvention. When specific classes are stated, this is understood toidentify any gene or enzyme within the specific classification. CIVPS orinteins are protein sequences internal or adjacent to a parent proteinsequence, that may spontaneously cleave themselves at either, or both,the carboxyl or amino terminal ends and are capable of selectivelyligating the resulting extein protein fragments when appropriate, underspecific conditions. See, for example, Perler, et al., Nucl. Acids Res.,22:1125-1127 (1994); Wallace, C. J., Protein Sci., 2:697-705 (1993); Xu,et al., Cell, 75: 1371-1377 (1993); Pietrokovski, S., Protein Sci.,2:697-705 (1994). Thus, CIVPSs may be said to be in-frame, self-cleavingpeptides that generally occur as part of a larger precursor proteinmolecule. CIVPS or inteins differ from other proteases or zymogens inseveral fundamental ways. Unlike proteases that cleave themselves orother proteins into multiple, unligated polypeptides, inteins have theability to both cleave and ligate in either cis or trans conformations.Thus as opposed to terminal cleavage that would result from the reactionof a protease on a protein, inteins have the ability to cleave atmultiple sites, and ligate the resulting protein fragments. Thiscleavage is induced under specific conditions and may be brought aboutimplementing techniques that are known in molecular biology. Inteinsfrom various sources, their sequences, characteristics and functionshave been described fully in the literature. See, for example, Kane et.al., Science 250:651 (1990); Hirata et al., J. Bio. Chem. 265:6726(1990) (Sacchromyces cerevisiae); Davis et al., J. Bact. 173:5653(1991), Davis et al., Cell 71:1 (1992) (Mycobacterium tuberculosis);Perler, et al., PNAS 89:5577 (1992) (Thermococcus litoralis). As shownin FIG. 1, the combination of an CIVPS with a protein of purportedactivity or structural role yields an intein modified protein, whosepurported activity or structural role may be substantially altered.Transgenic plants that express CIVPS modified proteins (from theirassociated intein modified genes) are an improvement upon previoustransgenic plants, because the parent intein modified protein can havetwo substantially different states that are controllably mediated byintein cleavage. This cleavage may or may not be associated withrecombination of the purported protein sequence. The invention may beformed from any plant species, combined with any combination of singleor multiple proteins and CIVPS. Plant species may include, but are notlimited to: poplar, birch, cedar, pine, hardwoods, softwoods, soybeans,switchgrass, corn, tobacco, alfalfa, sugar cane, cauliflowers,artichokes, bananas, apples, cherries, cranberries, cucumbers, lettuce,grapes, lemons, melons, nuts, tangerines, rice, oranges, peaches, pears,blueberries, strawberries, tomatoes, carrots, cabbages, potatoes,endive, leeks, spinach, weeds, arrowroot, beets, carrots, cassava,turnips, yams, radishes, sweet potatoes, wheat, barley, soya, beans,rapeseed, millet, sunflower, oats, peas, tubers, bamboo, seaweed, algae,or any other plant species. Proteins may include any known, putative,modified, or de novo created proteins. Although the selection of thenative protein is not restricted, preferred proteins includelignocellulosic degrading proteins (cellulases, lignases, amylases),starch degrading enzymes (amylases, glucanases), enzymes in thebiosynthetic pathways required for fuel or chemical production,bacterial or viral antigens, enzymes in the biosynthetic pathways forvitamins or other food additives (phytases, cellulases, amylases,glucanases, hemi-cellulase, pectinase, protease, xylanase, lipase,growth hormone), proteins that impart pest or insect resistance,proteins that impart herbicide resistance, and therapeutic proteins(insulin, erythropoietin, growth hormone, leptin, tissue plasminogenactivator, tumor necrosis factor receptor, Her2 receptor) implicated indisease pathogenesis. The choice of CIVPS or intein used to modify theprotein, the fusion of which is expressed in the desired plant, is alsonot limited. Any single or multiple CIVPS or intein may be used in anyconfiguration with respect to the desired protein or proteins. The CIVPSor inteins should have the capability to be spliced at one or both endsin response to some stimuli, and may or may not permit ligation of theproteins to which single or multiple CIVPS or inteins are fused.

Transgenic plants expressing CIVPS or intein modified proteins, and theproduction of CIVPS or intein modified proteins in transgenic plants canbe accomplished by combining methods (Ausubel, et al.) known in the art.Generally, these methods include construction of a DNA containing theCIVPS or intein modified protein of interest and the necessaryregulatory elements required for its expression, amplification andselection of the constructed DNA, transformation of the desired plantspecies, regeneration and selection of the appropriately transformedplant species, and if necessary, purification of the CIVPS or inteinmodified protein in its native form or the cleaved form. Both theproduction of transgenic plants expressing CIVPS or intein modifiedproteins, and the production of CIVPS or intein modified proteins intransgenic plants form part of this invention. For the production of thetransgenic plants, or CIVPS or intein modified proteins in transgenicplants, the CIVPS or intein modified protein DNA sequence must beconstructed. This is easily accomplished by cloning the gene sequence ofthe desired activity and the desired intein sequence into E. coli or anyother suitable host (e.g., yeast may be beneficial in some cases, orexpression in mammalian or plant cells with or without the use of viralor non-viral vectors). Once the gene and intein coding sequences havebeen cloned, they must be joined in the desired configuration. Thechosen intein sequence should be able to perform the desired functionssuch as splicing in response to an imposed stimuli (for example, light,pH change, temperature, pressure, or changes in the local chemicalcomposition surrounding the intein modified protein), and if necessarypermitting ligation of the fused protein. Joining of the CIVPS orintein's DNA sequence and the protein's DNA sequence is easilyaccomplished by methods known in the art, resulting in CIVPS or inteinmodified protein DNA coding sequences, or combinations thereof, as shownin FIG. 1. As already indicated, an CIVPS or intein modified protein isone which fuses the CIVPS or intein to one of either the carboxyterminal, amino terminal, or internal portions of the native protein orproteins. Although many alternative methods exist, one way of creatingthe fusion between the CIVPS or intein and desired protein codingsequences would be to purify the DNA encoding the desired proteinsequence, use a restriction enzyme to cut the protein coding sequence atthe desired point of intein insertion, and then ligate the intein codingsequence into the restricted site. The polynucleotide, or either of thenucleic acid segments may be cloned directly to appropriate regulatoryand/or selection sequences, or via a vector them. Examples of regulatorysegments are promoters to control the temporal expression of the CIVPSor intein-modified protein, origins of replication, and/or signalingsequences to control the spatial distribution of CIVPS orintein-modified proteins in vivo in specific plant tissues and/orspecific subcellular compartments, and/examples of selection elementsare herbicidal or antibacterial genes, fluorescent makers, dye markers,and other suitable selective markers. The resulting polynucleotide orvector comprising the CIVPS or intein modified protein(s) encodingpolynucleotide(s), and optionally any desired regulatory, and selectionelements, then may be amplified to obtain larger amounts of product,which may be used for subsequent transformation of a desired plantspecies. Modification of any and all of these steps is possible tofacilitate specific orientation and fusion between any desired CIVPS orintein(s) and protein(s) polynucleotides, and it is conducted employingmethods that are known in the art. Alteration of either the codingsequences and/or the CIVPS or intein coding sequence and the ligation ofeither or both of these sequences may also be easily accomplished bytechniques known in the art, such as site-directed mutagenesis, codonoptimization, random mutagenesis, polymerase chain reaction (PCR),error-prone PCR, and/or any other suitable method that would beconsidered routine by an artisan. These techniques facilitate theplacement of a number of joining sequences, and any desirable andsuitable combination may be used. Likewise, any combination ororientation of regulatory and selective elements may also be implementedin accordance with this invention. Gene regulatory elements, such aspromoters (Guilley et al., Higgins, T. J. V., Coruzzi et al., Tingey etal., Ryan et al., Rocha-Sosa et al., Wenzler et al., Bird et al.),enhancers (Brederode, et al.), RNA splicing sites, ribosomal bindingsites, glycosylation sites, protein splicing sites, subcellularsignalling sequences (Smeekens et al., van den Broeck et al., Schreieret al., Tague et al.), secretory signal sequences (Von Heijne, G.,Sijmons, et al.), or others may be advantageous in controlling eitherthe temporal or spatial distribution of the CIVPS or intein modifiedprotein concentration and activity in vivo in the transformed plant. Useof these elements may be desired to facilitate the production andprocessing of intein modified proteins in transgenic plants. Theexpression of the intein-modified protein(s) may be conducted either ina constitutive or induced manner. In order to attain either of thesemodes, any of the methods that are either described in this patent orknown in the art, or later made available, may be implemented. Theinduction of protein expression may be attained with the aid of aforeign stimulus(i). Examples of these are the exposure to apesticide(s), to light, a temperature change(s), and/or sound(s). Otherforeign stimuli, however, may also be employed. In addition, therecombinant plant may also express any one or more of the selectablemarker gene or reporter gene(s) mentioned above.

Once the CIVPS or intein modified protein DNA sequence has beenconstructed, optionally codon optimized, combined with the desiredregulatory and selection DNA sequences, successfully cloned andselected, then transformation of the desired plant species andgeneration of full plants is required. Methods for transformation of adesired plant species, and the generation of full plants can beaccomplished by techniques known in the art (Draper, et al., Potrykus,et al.). Transformation techniques include, but are not limited to:Agrobacterium tumefaciens mediated gene transfer, Agrobacteriumrhizogenes mediated gene transfer, direct gene transfer to plantprotoplasts, Ti plasmid mediated gene transfer (with or without a helperplasmid), biolistic or particle bombardment plant tranformation(Gordon-Kamm et al.), microinjection and fiber-mediated transformation,and tissue electroploration (Shimamoto et al.). Gene transfer may occurin whole plants, plant explants (such as, but not limited to rootexplants), any plant portion (such as, but not limited to plant leafsegments, seeds, or seed segments), plant protoplasts or apoplasts, orsingle or multiple plant cells. Each different method has beensubstantially described in detail by the prior art. Methods of selectionof properly transformed plants are known in the art. Selection methodsmay be facilitated by including a selectable marker in the transformedDNA containing the CIVPS or intein modified protein (such as aresistance gene, gene coding the production of a colored compound, genecoding the production of a fluorescent compound, or any other suitablemethod). Additionally, DNA from transformed plants may be isolated andsequenced to confirm the presence of the desired CIVPS or inteinmodified protein coding sequence. Other techniques are also suitable forconfirmation of the selection process, such as polymerase chainreaction, restriction digest analysis and southern analysis. Any methodof selection that allows identification of the desired transgenic plantmay be used. Once the plant is transformed with the CIVPS or inteinmodified protein and desired regulatory and selection sequences, wholeplants can be regenerated by methods know to the art (Horsch et al.).Most methods consist of culturing the transformed plant cells, explants,tissues, parts, or whole plants in the proper medium and underappropriate conditions of light and temperature. The method used toregenerate the plant should not limit the invention and any effectivemethod may be used. The resulting transgenic plant should produce CIVPSor intein-modified proteins that are substantially described as, or acombination of, those shown schematically in FIG. 2. Once the whole,transgenic plant has been selected, it can be monitored for CIVPS orintein modified protein expression. This is not required for theproduction of transgenic plants expressing CIVPS or intein modifiedproteins, but is prudent to confirm that the desired transgenic plantexpressing the desired CIVPS or intein modified protein has beenobtained and expression is properly controlled by the desired controlelements used. Monitoring of CIVPS or intein modified protein expressionis necessary for the purification of the CIVPS or intein modifiedproteins in the cleaved or uncleaved state, as described schematicallyin FIG. 3 for either whole intein modified proteins, or components ofintein modified proteins that are composed of combinations of elementsshown in FIG. 3. Protein expression of the intein modified protein canbe monitored by western analysis, 2-dimensional gel electrophoresis (andstaining), or mass spectrometry, conducted on plant extracts or proteinfractions purified from the transgenic plant. In addition, either someof the purified proteins, or the transgenic plant itself, should beexposed to the intein cleavage stimulus. After exposure, both the CIVPSor intein modified protein and the resulting protein that appears as aconsequence of CIVPS or intein cleavage can both be analyzed by westernanalysis, and other assays, to verify the presence of the appropriateproteins, and the difference in activity between the intein modifiedprotein and the resulting cleaved protein. The activity assays must bedesigned so as to monitor the desired protein activity and should bespecific to that activity and not vulnerable to competing interferences.A control can be used as a standard to compare the native activity withboth the intein modified activity and the activity following inteincleavage. Methods and processes using transgenic plants expressing CIVPSor intein modified proteins include the use of the plants as substratesfor fuel production (including, but not limited to: burnable biomass,ethanol, methanol, propanol, propane, methane, or octane production),the use of the plants as substrates for commodity chemical production(including, but not limited to: lactic acid, ethanol, glucose or otherhexoses, pentoses, propane diols, ethene, ethane, ethylene, phenoliccompounds, amino acids, paper pulp, pesticides, insecticides, otheralcohols, other ethers, other esters), the use of the plants assubstrates for food production and or food additive production(including but not limited to: amino acids, sugars, vitamins, fiber, orcattle feed), the use of the plants for vaccine delivery, the use of theplants for the production of therapeutic proteins (including but notlimited to: insulin, erythropoietin, growth hormone, leptin, tumornecrosis factor receptor, glucagon, gamma interferon, or Her2 receptor),the use of the plants for paper production, and the use of the plantsfor remediation of waste materials. Any batch, semi-batch, or continuousprocess in which transgenic plants that express intein modified proteinsare used as substrates for one of the purposes described above isclaimed. These processes may include, but are not limited in scope to,processes in which the transgenic plants expressing intein modifiedproteins are harvested, exerted to the intein cleavage stimuli, mixedwith other substrates in a transgenic plant to substrate ratio greaterthan or equal to zero, and then converted either chemically,enzymatically, or biologically to one of the products detailed above.

The examples provided below illustrate the process of the invention, aswell as the manufacture of transgenic plants expressing CIVPS or inteinmodified cellulase enzymes, and the thus produced plants. In theseplants the cellulase enzymes are expressed as dictated by the regulatoryelements controlling the CIVPS or intein modified genes. The cellulaseactivity is substantially reduced in vivo by interruption of the nativecellulase enzyme by the fused intein. This allows the plant to grow,uninhibited or with little inhibition by cellulase activity. The plantsmay be harvested and exerted to the intein cleavage stimuli, such asexposure to a certain wavelength of light, mixed with a sulfurous or pHaltering chemical, mixed with a salt, mixed with any other chemical, orexerted to a change in temperature. In this case, the CIVPSs or inteinis be cleaved and the cellulase activity recovered, which then catalyzesthe cleavage of cellulose and/or lignin. At this point the cleavedprotein plant mash may be mixed in any proportion, preferably greaterthan or equal to zero, with other plant substrates, chemical substrates,municipal waste, manufacturing by-products, enzymes, and/or prokaryoticor eukaryotic cells, among others, to aid in the conversion of the plantsubstrate to the desired product, e.g. a fuel, commodity chemical, foodfor human or animal consumption, food additive, paper pulp, or vaccineantigen, among others. It should also be noted that the use of thepresent invention is not limited to manufacturing processes ormechanical processes. Non-limiting examples of applications of thisinvention are in the delivery of vaccines, hormones, or therapeuticproteins, in which case the intein modified protein may comprise acombination of therapeutic protein(s) and/or protein antigen(s),potentially protective protein sequences, and CIVPSs or intein(s) thatmay be expressed by the transgenic plant, e.g. a banana plant. Thedelivery process may occur, for example, by ingestion of the plantproduct by a human or non-human animal. The plant is then masticated inthe mouth and exposed to a stimulus(i) in vivo in the stomach, which inturn triggers or induces cleavage by the CIVPS or intein. In the case ofhumans the stimulus may be the reduced pH of the stomach, which inducesthe cleavage of the CIVPS or intein from the antigen or therapeuticprotein, and provides for appropriate ligation, if necessary. Thetherapeutic protein or antigen would then flow into the duodenum, orsmall intestine, where the pH would be neutralized and protein productsare now ready to be absorbed into the blood stream.

Background For Exemplary Information Provided Below

Many different variations in the protocol presented in Example 1 beloware suitable for practicing the present invention, as an artisan wouldknow. In general, a DNA sequence encoding a CIVPS or intein modifiedprotein is constructed and packaged into an appropriate vector, plantmaterial, whether it is single cells grown in suspension, protoplasts,plant segments or parts, whole plants, or other forms suitably describedhere are transformed with the vector, and complete plants, seeds, orother plant forms described here are regenerated. Example 1 shows oneembodiment of the inventive method, variations of which are possiblethat may be used to generate a transgenic tree, e.g. a poplar speciesexpressing an intein modified cellulase. The choice of desired protein,however, depends upon the application the transgenic plant species isintended for. In this regard native proteins, de novo syntheticproteins, or evolved proteins, e.g., by gene shuffling, error prone PCR,or any other analogous method, may be used. Cellulases catalyze acleavage reaction in breaking down cellulose, a chemical component ofthe plant. While other plants have been constructed expressingcellulases the enzymes typically have to be transiently expressed, orsequestered in parts of the cell so as not to disrupt plant tissuedifferentiation and development. See, for example, Ziegler et al.(2000); Dai et al. (a), (2000); Dai et al. (b) (2000);Montalvo-Rodriguez et al. (2000). Hence, in the case where the cellulaseactivity is not controlled by localization or transient expression,whole plants are often very difficult to regenerate, or the cellulaseactivity is often too low to be useful. By using an intein modifiedcellulase, the whole plant can be regenerated while the less activateintein modified cellulase is produced throughout the plant and at hightiter. See, Aspergen et al., Molecular Breeding 1:91-99 (1995). Theenzyme can be subsequently activated by the self-splicing ability of theintein to yield a cellulase of increased activity relative to the inteinmodified cellulase. It is noteworthy that any native protein will meetthe requirement for this invention, and selection of the protein isdependent upon the plant's intended purpose. In this case, a poplarspecies that could be induced to de-polymerize its own cellulose wouldbe beneficial for ethanol production from biomass, or as a substrate forfermentation of other chemicals.

Contruction of CIVPS or Intein Modified Proteins

Various recombinant DNA techniques may be used in combination toconstruct the vector carrying the DNA encoding the modified protein. Oneof the easiest and most direct utilizes the polymerase chain reaction(PCR) to assemble the nucleic acid sequence encoding the intein-modifiedprotein with appropriate complementary ends that facilitates ligationinto the desired vector. The PCR method is illustrated here. Othermethods may be used to accomplish this same goal, and some rely onspecific restriction and ligation of the desired protein and inteinencoding sequences, but may still include PCR steps. PCR Kits forconducting the reaction are readily available (Epicentre, Madison,Wis.). The only requirements on the primers is that one should match the5′ end of the sense strain to be amplified, and the other should matchthe 5′ end of the corresponding antisense strain; relative sequenceuniqueness is beneficial.

Clean-Up and Purification from a Gel

The purification of DNA from a gel may be accomplished usingelectroelution, phenol extraction, agarase digestion, glass beadextraction, or from a number of commercially available kits. Thecommercially available QIAquick Gel Extraction Kit (Qiagen, Valencia,CA) and associated method is one example.

Selection of Intein According to Intended Use

Two features are of importance in this step: the property the CIVPC orintein possesses to induce splicing that will facilitate optimization ofthe transgenic plant for its intended purpose, and where to place theintein within the nucleic acid sequence encoding the target protein. Anycoding sequence for a self-splicing protein, i.e. an intein, may be usedin this invention. A compilation of some known inteins is given on thefollowing website http://blocks.fhcrc.org/˜pietro/inteins/. Otherinteins remain to be discovered and new inteins may be created throughsequence analysis, recombinant DNA methods, and mutation of knownsequences. This intein of Example 1 is advantageous for the intendedtransgenic poplar species because upon splicing it yields predominantlyligated, native protein (>75%), and is temperature sensitive so thatintein splicing is inhibited at temperatures less than 30° C., and isnot substantial until 50° C., at which temperature the half-life of theuncleaved protein is less than 2 hours.

Construction of Intein Modified Protein

In order to ensure proper intein splicing, the intein is inserted inExample 1 in frame next to a serine, cysteine, or threonine residue ofthe native target protein. This leaves the native target protein'sserine, cysteine, or threonine on the carboxylic acid side, of thisintein's histidine—asparagine, conserved residues at the terminal 536and 537 intein amino acid positions, respectively. Other terminalresidues may be used, depending upon the desired stimulus and mechanismfor intein splicing. If desired, the codons at the extein-inteinjunction may be altered to facilitate these requirements. Care isadvised when altering the junction codons, so that the intein modifiedprotein may cleave as desired, and allow the resulting products toperform the appropriate activity. The intein position within the nativetarget protein such is to substantially change the activity of theresulting intein modified protein. In most circumstances virtually anyinterruption within or near the active site of the molecule meets thiscriterion. The combination of the amplified intein sequence and theamplified native protein sequence is easily accomplished if a serineresidue resides close to a unique restriction site of the nativeprotein's coding sequence. Conversely, the intein coding sequence isreadily incorporated at any desired position in the native proteinsequence by using several polymerase chain reactions. A preferred PCRmethod is set forth here. Preferrably 50 oligonucleotide primers areused. Shorter primers may be used, however it is beneficial, althoughnot necessary, to use primers of the same length. The sense primer ofthe C-extein may hybridize to both the C-extein and intein sequence atthe junction to facilitate the fusion of the amplified sequences insubsequent PCR amplifications. For intein amplification, both primerspreferably overlap with their respective desired adjacent exteinsequences to facilitate fusion of the intein sequence and exteinsequences in subsequent PCR amplifications. The polymerase chainreaction is preferably carried out using the standard protocol outlinedabove, but may have some optimization. Typical optimization parametersare the amount of template and primer DNA added to the mixture(generally the primer DNA is added in great excess relative to thetemplate DNA), the temperatures and times for the reaction cycles, thenumber of cycles, and the MgCl₂ concentration. The length andcomposition of the primers used may also be varied to yield an effectiveintein modified protein, so long as the constraints on placement areobserved. Kits are commercially available which include all necessaryreagents: Taq DNA polymerase, MgCl₂, 25 mM dNTP mixture (containingequamolar amounts of dATP, dCTP, dGTP, and dTTP), reaction buffer, andwater.

At this point the next round of PCR is started to fuse the extein andintein sequences. In this case the intein fragment is preferably mixedwith an equimolar portion of the C-extein cellulase fragment.Combination of these fragments represents both the template and primers(overlapping regions) to be used. Addition of reaction buffer, 25 mMdNTPs, MgCl₂, and Taq DNA polymerase is still required, as are thechanging temperature cycles. This reaction is preferably augmented byaddition of the following sense and anti-sense primers, respectively,along with E. coli DNA ligase (New England Biolabs, Beverly, Mass.),however this addition is not necessary and depending upon the exactreaction conditions employed may not lead to an increase in the yield.5′-ACAGAATGGGGAACGAGCGATGCTAGCATTTTACCGGAAGAATGGGTTC-3′ [SEQ ID NO: 1]5′-CGTGTCTGCTCCGTTTACCGCTTTTTTTAATTGGACGAATTTGTGCGTGA-3′ [SEQ ID NO: 2]

Once completed, the PCR products are preferably again run on an agarosegel, and the appropriate band, 2665 nucleotides long, purified from thegel and analyzed according to the methods described above. A smallamount of the purified reaction product is preferably used forquantitation by measuring the absorbance at 260 nm and 280 nmwavelengths on a UV spectrophotometer. To complete assembly a PCRreaction of the intein modified cellulase coding sequence is carried outcombining equimolar amounts of the fused C-extein and intein fragmentsjust constructed, with the N-extein fragment purified previously. ThePCR reaction is preferably conducted using the same temperature cyclesas in the previous reaction after addition of reaction buffer, 25 mMdNTPs, MgCl₂, and Taq DNA polymerase. This reaction is preferablyaugmented by addition of the following sense and anti-sense primers, andE. coli DNA ligase (New England Biolabs, Beverly, Mass.); however thisaddition is not necessary and depending upon the exact reactionconditions employed may not lead to an increase in the yield.5′-AGCATTCAGACCTCCCATTTCATACGAAAAGAGGAAATAGATAGATTTTC-3′ [SEQ ID NO: 3]5′-CGTGTCTGCTCCGTTTACCGCTTTTTTTAATTGGACGAATTTGTGCGTGA-3′ [SEQ ID NO: 4]

Vector Construction

Other elements may be included in the expression cassette prepared inExample 1, e.g. extracellular secretion signaling sequences,intracellular localization signaling sequences, other induciblepromoters, etc. As the vector is now contained within the recombinantstrain A. tumefaciens, the gene transfer to the poplar plant relies onthe bacteria's specialized delivery system. Other gene transfer methodsare available, and selection of a suitable transformation method dependsupon the source of the plant material. For example, protoplasts orindividual plant cells may be transformed directly with the recombinantpTiBo542 plasmid using electroploration, calcium chloride, or Biolisticparticle bombardment (Bio-Rad, Hercules, Calif.). Conversely plantcallus, plant segments, or in some cases, whole plants may be used asstarting material, when appropriate. For efficient gene transfer tooccur, the time of incubation and cell density of the culture ispreferably optimized.

Advantages and Uses for Transgenic Poplar of Example 1

The resulting transgenic poplar species may be grown and passagedindefinitely while producing the intein modified cellulase in hightiter. The cellulase may be subsequently activated by harvesting theplant, mechanically chipping or grinding it to increase the exposedsurface area, and then incubating the resulting mash in a vat or tank atan elevated temperature (preferably 30° C. to 50° C.) and/or lower pH(6.5 or below). Exposure to the elevated temperature, and lower pH, ifused, will induce the intein splicing and yield the native cellulase ata substantially increased activity. Under these conditions the cellulasemay now catalyze the cleavage reaction of cellulose to economicallyproduce substrates that may be subsequently fermented into ethanol orother chemical entities. In addition, this plant may be used as a sourceof either the intein modified cellulase, or the recovered nativecellulase, post splicing. In either case, the protein is preferablypurified from the plant using methods well known in the prior art, suchas precipitation, membrane filtration, chromatography including affinitychromatography, extraction, etc.

The use of transgenic plants producing intein modified proteins has twoadvantages over previously reported transgenic plants. Because theintein modified protein has substantially less activity than the nativeprotein, it may be expressed at higher titer and localized anywhere inthe plant species. Previous reports of transgenic plants expressingcellulase enzymes have taught elimination of the secretion signals tocontain the cellulase enzymes in the cytosol of cells. This is notnecessary with the use of intein modified proteins and is a substantialimprovement as the modified protein may be placed in close proximitywith its substrate, but not catalyze the reaction until desired. Inaddition, these plants have a higher degree of environmental safety.Because the genes transferred encode proteins of substantially lessactivity under physiological conditions, horizontal gene transferbetween species is less likely to impart any selective advantage. Forthis reason it is unlikely that either the transgenic plants wouldoutperform native plants in the wild, or that gene transfer would yielda selective advantage favoring a transformed population.

Example 2 demonstrates the broad applications of this invention. Example2 shows a variation of the method of Example 1 to generate a transgenicDouglas-fir species expressing an intein modified lignin peroxidase. Thechoice of a specific target protein depends upon the applicationintended for the transgenic plant species. For this example, a ligninperoxidase gene that facilitates the catalytic breakdown of lignin, achemical component of wood was selected. By using an intein modifiedlignin peroxidase, the whole plant may be regenerated while theinactivated intein modified lignin peroxidase is produced throughout theplant, at high titer if desirable. The enzyme may be subsequentlyactivated by the self-splicing ability of the intein to yield the nativelignin peroxidase at increased activity than the intein modified ligninperoxidase. This allows improved control of the lignin peroxidaseactivity that is not currently available. Such a transgenic plantspecies is valuable for the production of pulp, animal feeds, substratesfor other processes, improvements on mechanical pulping, biobleaching ofpulp, improvement from decreased pulp processing wastes, and theproduction of biopolymers with unique properties.

Construction of Gene & Intein Modified Protein

As indicated above, any native protein is suitable as the targetprotein, and its selection is dependent upon the plant's intendedpurpose. For this example, a Douglas-fir species that may modify its ownlignin is beneficial as a substrate for different pulping processes. Theprotein encoding nucleic acid of interest may be isolated fromPhanerochaete chrysosporium (GenBank Accession #M37701). One primerpreferably matches the 5′ end of the sense strain to be amplified, andthe other the 5′ end of the complementing DNA strand at the end of thegene,It is beneficial to have relative sequence uniqueness.

Purification of PCR Fragments from Gel

The purification of the nucleic acid from the gel is accomplished usingelectroelution, phenol extraction, agarase digestion, glass beadextraction, or from a number of commercially available kits. Preferablythe commercially available QIAquick Gel Extraction Kit, available fromQiagen (Valencia, Calif.) is used.

Intein Selection

The choice of intein is very dependent upon both the intended purpose ofthe plant and the intein modified protein. Many different inteins existand may be used. For this example an intein with the same properties asin Example 1 is beneficial for the intended use of a transgenicDouglas-fir species. Hence, a variant of the Psp pol intein (GenBankAccession #PSU00707) from Pyrrococcus spp. is preferably used. Theadvantage of this intein is that upon splicing it yields predominantlyligated, native protein (>75%), and is temperature sensitive so thatintein splicing is inhibited at temperatures less than 30° C., and isnot substantial until 50° C., where the half-life of the uncleavedprotein is less than 2 hours. This intein induces splicing in vitro by apH shift, thus adding increased flexibility to subsequent processing ofthe transgenic plant.

Vector Transformation

With the vector contained within the recombinant strain of A.tumefaciens, gene transfer to Douglas-fir relies on the bacteria'sspecialized delivery system. Other gene transfer methods are available,and selection of a suitable transformation method depends upon thesource of the plant material and ease with which the method can beapplied. Some modification and optimization of the transformationparameters is usually necessary.

Uses of the Recombinant Trees

The tree of Example 2 may be used as a source material for thepurification of lignin peroxidase or intein-modified lignin peroxidase.Alternatively, it may be used also by itself as a substrate forproducing wood pulp in any number of applications, e.g. paperproduction, animal feed, composite materials, etc. Both Example 1 andExample 2 have illustrated the use of trees, certainly other plants areuseful options and depend upon the intended use of the invention. Inmany areas these types of trees do not grow well and grasses, vines,seaweed, or other plant species do, and may be used equally well. Inaddition, many fruits and vegetables may benefit from intein modifiedprotein technology, such as for example to induce ripening, pesticideresistance, or any number of other applications. Hence the choice ofhost plant is not limiting. The use of plants as sources for recombinantproteins is facilitated by use of the CIVPS or intein technology of thisinvention. Plants are made to express any number of fusion proteinswhere the fusion point is comprised of an intein that does notfacilitate recombination of fused protein exteins, but instead links thedesired protein to a binding protein for purification via affinitychromatography. In this case the desired protein may or may not havefull activity in vivo. Once expressed in the plant, the fusion proteinis eluted onto an affinity column where the binding portion of thefusion protein binds the column. The column is then treated to induceintein splicing and the desired protein is washed away and recovered.Another variation of the invention that is of medical interest is afusion protein comprising a therapeutic protein or vaccine fused byinteins to protect protein groups or relying on inteins to distrupttheir natural activities when in the plant. Such a therapeutic proteincan be expressed in a plant and purified and injected, or simply eatenby a human and non-human animal, e. g. in the case of animal vaccinationor hormonal treatment. Intein splicing then occurs either in vitro in aprocess tank or inside the patient, or animal, relying on the change ofpH within the stomach, or a thiol gradient induced by ingestion of athird chemical. Splicing removes the protective protein groups, yieldingthe native therapeutic protein or vaccine, which is then absorbed in thegut.

Either of the transgenic trees expressing intein modified proteins fromExamples 1 and 2 may be effectively used in an industrial scale processas is shown in Example 3. The pulping itself may be enhanced by amodification similar to that used in Example 2 for the Douglas-firspecies.

Tree Processing

Typical pretreatment processes for the degradation of lignocellulosicsubstrates include concentrated acid pretreatment (usually SulfuricAcid), dilute acid pretreatment, ammonia explosion pretreatment, and hotwater pretreatment. Other pretreatment processes are possible, anddesign of the transgenic tree expressing an intein modified proteinshould be optimized to take full advantage of a pretreatment processwhen necessary. Intein splicing may occur in a vessel via any knownmethod, such as, but not limited to: pH shift, temperature change, lightexposure, acoustic stimulation, or any exogeneous chemical addition.

Intended Uses and Process Variations

Preferred variations of the process of Example 3 include combining thepretreatment, splicing, digestion, and fermentation steps. Thispreferred processing consolidation may occur between any of the steps,however a preferred manifestation incorporates all steps simultaneouslyin a single unit operation. This preferred combination may realize costsavings through a decrease in capital expenditure and depreciation,decrease in the cost of substrates, and a process dependent decrease inthe cost of energy and chemicals input to the process. In addition, asopposed to competing chemical processes making the same products,environmental benefits may be realized through decreased emissions andhazardous waste generation. In Example 3 the choice of product isdependent upon the organism used in the fermentation for the desiredbioconversion. Any organism that may adequately utilize the degradedcellulose as a substrate may be used to effectively produce a desiredproduct. For this reason the spectrum of end products that may be madeis very large. Applications that will benefit from substrates withpreferred processing traits facilitated by the intein modified proteinscarrying plant of this invention include, but are not limited to, fuelproduction, chemicals production, textiles production, biopolymerproduction, food production, and saccharification. Although Example 3 ismostly focused on the fermentation of the degraded transgenic plants,intein modified plants may also be used as substrates for traditionalchemical processes. For example, the plants of Example 2 may bepreferentially used in paper pulping. In such a process, benefits arederived from a decrease in the harsh chemicals used to bleach the wood.This will likely result in a decrease in the costs of chemical input,hazardous material generation and containment, and potentially someconsolidation in processing. Another use is that of pectinase for cottonscouring, or cellulases for other textile production processes. In theseinstances, the end products are derived from more traditional chemicalprocesses, although benefits accrue through the use of intein modifiedprotein plant substrates, as opposed to the normally harsh chemicalprocessing environment generally employed.

Animal feed is commonly supplemented with a variety of enzymes used toincrease the nutritional value of the feed, as well as decrease theenvironmental burden experienced in proximities where animal manureaccumulation is substantial. Nutritional value is increased through theputative enzyme action on plant polymers, which assist the animal indigesting the feed and thereby utilizing more of the beneficial feedcomponents. The environmental burden may be decreased by limiting theamounts of added minerals, such as inorganic phosphate, which may beobtained from the plants themselves in the presence of the activeenzyme. The benefits associated with using intein modified proteins, asopposed to unmodified proteins, result in multi-protein expression, athigh levels, which do not interfere with plant regeneration yet impart adesired enzyme activity upon splicing within the animals stomach. Thisdecreases the cost of feed by delivering the enzymes within the mealitself, as opposed to their being produced exogenously and added to themeal. In addition, the added benefit of using genes that code for nearlyinactive proteins in vivo in plants, provides a technology platform thatis less likely to be associated with environmental risks associated withhorizontal gene transfer to native plant species. This advantageousenvironmental affect, whether real or perceived, holds for all inteinmodified protein plant products. Example 4 illustrates the constructionan intein modified phytase in rapeseed, for use as animal feed.

Uses and Variations

Phytase is an enzyme that assists in the evolution of inorganicphosphorous from myoinositol phosphates contained inherently in animalfood. An economic impact is brought about through a decrease in theamount of phosphate supplementation required for the production ofanimal feed, and a decrease in the phosphate content of the animal'smanure, which contributes to the contamination of local waters. AlthoughExample 4 below illustrates the construction and use of an inteinmodified phytase expressed in rapeseed for animal feed, a number ofother valuable native proteins may be used as well. For example, phytasemay be substituted with, or used in addition to any number ofcellulases, amylases, glucanases, hemi-cellulases, pectinases,proteases, xylanases, lipases, growth hormones, or immunogenic antigens,among others. Each of these other proteins has a potential economicvalue in the use of animal feed supplementation.

Example 5 illustrates one of the preferred embodiments of the invention.A transgenic corn is constructed and used as a substrate for ethanolprocessing. In this case the intein modified gene sequence of Example 1is again used for demonstration purposes only. In a preferredembodiment, however, several intein modified proteins may be expressedsimultaneously to optimize the desired plant degradation processingtrait for use in the fermentation process. The target enzymes may beselected from enzymes commonly known as cellulases (E.C. 3.2.1.4),exocellobiohydrolases (E.C. 3.2.1.91), glucosidases (E.C. 3.2.1.21), andmay be expressed optimally with other enzymes selected from the EnzymeClassification heading 3.2.1.x, or any other classification groupnecessary. In addition to the simultaneous expression of multiple inteinmodified proteins, the preferred composition of matter embodiment is afertile plant capable of reproduction and stable gene inheritance.

Transformation Information

The macroprojectiles are used to accelerate the microprojectiles, whichenter the plant cells.

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples, which are includedherein for purposes of illustration only, and are not intended to belimiting of the invention or any embodiment thereof, unless and where itis so specified.

EXAMPLES Example 1 Production of Transgenic Poplar Expressing anIntein-Modified Cellulase

For this example a cellulase enzyme is used. A vector is first assembledcontaining the DNA coding sequence for the intein modified protein. Inorder to construct such a vector, an intein modified protein DNAsequence is first prepared, and then packaged into the desired vector.The desired protein for this plant is a cellulase (GenBank Accession#AY039744) isolated from Bacillus sp. NBL420. The gene corresponding tothis protein is amplified using PCR from a genomic DNA template isolatedfrom the Bacillus sp. NBL420. The PCR reaction is performed by mixingthe template DNA, two primers complimentary to the 3′ ends of thetemplate DNA to be amplified, Taq DNA polyermase, reaction buffer (500mM KCl, 100 mM Tris-Cl pH 9.0, 0.1% Triton X-100), and MgCl₂ in athin-walled 250 μL PCR tube. Once mixed, each reaction tube is placed ina thermocycler, and the thermocylcer is set for 35 cycles comprised ofthree segments: 94° C. for 30 seconds, 60° C. for 60 seconds, 72° C. for120 seconds. Following amplification, the resulting PCR product isanalyzed by electrophoresis on a 1% agarose gel, along with molecularweight standards (Invitrogen, Carlsbad, Calif.), with the aid of 1×TAE(or TBE) running buffer, and stained with ethidium bromide (0.5 μg/mL).Care should be taken to ensure that the appropriately sized band, ofapproximately 3200 base pairs (bp), has been obtained. This band is thencut out from the gel with a scalpel, and purified (separated from thegel material) using a commerically available gel purification kit(Qiagen). Once the fragment has been purified from the gel, the band isanalyzed using restriction digestion or sequencing as described byAusubel et. al., Current Protocols in Molecular Biology, Wiley, N.Y.(1998). After gene amplification, the gene is modified by insertion ofthe intein sequence segment. In this case, a variant of the Psp polintein (GenBank Accession #PSU00707) from Pyrrococcus spp. is used Thisvariant, described in the literature, contains a mutation at thetyrosine 534 residue which converts that tyrosine to methionine. See,Xu, M, Perler, F, (1996), The mechanism of protein splicing and itsmodulation by mutation, The EMBO Journal 15:5146-5153. This intein maybe cleaved in vitro by a pH shift. The coding sequence of this intein isthen amplified by PCR using genomic DNA from Pyrrococcus spp, as atemplate. The PCR reaction is conducted using a standard protocol (e.g.,30 cycles comprised of a 94° C. for 30 seconds, 50° C. for 60 seconds,72° C. for 120 seconds) and the following primers.5′-ATTATGTGCATAGAGGAATCCAAAG-3′ [SEQ ID NO: 5]5′-AGCATTTTACCGGAAGAATGGGTTC-3′ [SEQ ID NO: 6]

Once the amplification is complete, the PCR product is transferred toand elecrophoresed on a 1% agarose gel in 1×TAE or TBE buffer. Theresulting band is then purified and analyzed as described above for thenative cellulase coding sequence. At this point the two PCR fragmentsshown in FIG. 1, one encoding the cellulase protein and one encoding theintein polypeptide sequence, are joined. Here, the intein is inserted inframe into the native protein, such that a serine residue of the nativeprotein becomes the terminal C-extein amino acid at the junction pointbetween the native intein and C-extein of the native protein. Thisintein modified protein segment is produced using PCR by firstamplifying the C-extein coding sequence of the cellulase gene. Primersthat overlap both the C-extein, and the intein end containing thehistidine and asparagine codons immediately adjacent to the C-extein areused to amplify the C-extein sequence:5′-CTTTGGATTCCTCTATGCACATAATTCCGGAAACGGCGGTGTCTACCTCG-3′ [SEQ ID NO: 7]5′-CGTGTCTGCTCCGTTTACCGCTTTTTTTAATTGGACGAATTTGTGCGTGA-3′ [SEQ ID NO: 8]

The resulting sequence is 604 nucleotides long. The intein is thenamplified using a sense primer that contains both the intein endcontaining the terminal serine codon, and the N-extein end of thecellulase gene, along with an antisense primer that contains specificnucleotides of the intein and C-extein. For this PCR reaction thefollowing primers are used to obtain a sequence 1661 nucleotides long:5′-ACAGAATGGGGAACGAGCGATGCTAGCATTTTACCGGAAGAATGGGTTC-3′ [SEQ ID NO: 9]5′-CGAGGTAGACACCGCCGTTTCCGGAATTATGTGCATAGAGGAATCCAAAG-3′ [SEQ ID NO: 10]

The N-extein is then amplified using PCR and one primer that containsspecific nucleotides of the sense N-extein strand, and another primerthat contains specific nucleotides of the N-extein and adjacent inteinsequence. The N-extein portion of the cellulase gene is amplified withthe following primers resulting in a sequence 1541 nucleotides long.5′-AGCATTCAGACCTCCCATTTCATACGAAAAGAGGAAATAGATAGATTTTC-3′ [SEQ ID NO: 11]5′-GAACCCATTCTTCCGGTAAAATGCTAGCATCGCTCGTTCCCCATTCTGTG-3′ [SEQ ID NO: 12]

Once these three reactions are complete, each PCR fragment is cleaned toremove residual primers, and the C-extein, intein, and N-extein PCRfragments are joined by conducting two more polymerase chain reactions.The intein and one of the cellulase extein regions, either the C-exteinor the N-extein, are amplified in a single reaction by mixing equimolarportions of the two PCR fragments generated above and performing PCR asdescribed earlier. This reaction requires no extra external primers andresults in the first intein-extein fusion. This reaction mixture iscleaned, and then equimolar portions of the cleaned fusion product aremixed with the remaining extein portion, and PCR is conducted once againwithout adding additional primers. No exogeneous primer is required ineither of the last two PCR reactions, and annealing occurs at theintein-extein junctions. The annealed regions are extended by Taqpolymerase resulting in the final fusion products. This sequence ofreactions results in the coding sequence of the intein modified proteinwith the intein inserted at the exact position desired. The product ofthe final reaction is cleaned again, and amplified using PCR one lasttime with primers specific to the cellulase extein termini with specificends to facilitate ligation into the cloning vector. Once this reactionis complete, the PCR products are run on an agarose gel, and theappropriate band, 3806 nucleotides long, is purified from the gel andanalyzed according to the methods described above. The resulting inteinmodified protein coding sequence (nucleic acid segment) contains aribosome binding site, a start codon at the beginning of the N-extein,the complete sequence of the intein modified cellulase with the inteininserted in frame in the proper orientation, and a stop codon at the endof the C-extein coding sequence. The intein modified cellulase codingsequence is then cloned into pTiBo542, replacing the tms and tmr genesin the T DNA, using the methods described in Ausubel, et. al., CurrentProtocols in Molecular Biology (1998). See, Parsons T J, Sinkar, V P,Stettler, R F, Nester, E W, Gordon, M P, “Transformation of Poplar byAgrobacterium tumefaciens,” Biotechnology 4:533-536, 1986. Here theexpression cassette includes a “MAC” promoter, a mannopine synthetaseterminator, and a kanamycin resistance marker. This vector istransformed into A. tumefaciens A281 using any suitable method known inthe art (e.g., electroploration, or the calcium chloride method).Various transformation methods are also described by Ausubel, et. al.(1998), above.

To transform the desired Populas trichocarpa x deltoides, H11 plantspecies, with the recombinant A. tumefaciens, a variation of the leafdisk method is employed. The recombinant A. tumefaciens is cultured inselective medium containing 50% MG medium (10 g/L mannitol, 2.32 g/Lsodium glutamate, 0.5 g/L KH₂PO₄, 0.2 g/L NaCl, 0.2 g/L MgSO₄-7H₂O,0.002 g/L biotin, pH 7.0), 50% luria broth (20 g/L tryptone, 10 g/Lyeast extract, and 10 g/L NaCl), and appropriate antibiotic, at 30° C.in an incubator-shaker. For plant transformation, small greenwood stemsections, approximately 7 mm in length and 2-3 mm in diameter, aresterilized with a 20% bleach, 0.1% Tween 20, and 30 mg/L Benomylsystemic fungicide (Chas. H. Lilly Co., Portland, Oreg.) solution. Afterwashing with sterile water, the stem sections are asepticallytransferred to a culture of A. tumefaciens at a cell concentration ofapproximately 5×10⁸ cells per mL, and the sections allowed to incubatefor 16 hours. After exposure to the recombinant A. tumefaciens culture,the plant stems are transferred to solid Murashige-Skoog mediumsupplemented with zeatin riboside and kanamycin in a vertical position.See, Murashige T, Skoog F, “A revised medium for rapid growth andbioassays with tobacco tissue cultures,” Physiol. Plant, 15:473-497,1962. Once roots have begun to grow, shoots will develop. Theregenerating plants are transferred to fresh plates every two to threeweeks, and a normal light cycle is maintained during plant growth and atelevated humidity in the incubator. Once roots form, the explants aretransferred to a solid medium lacking zeatin riboside, but containingkanamycin for another two to three weeks, after which period the plantsare transferred to boxes containing soil for four to five days prior toreplanting in soil and full growth in a greenhouse or controlled plot ofsoil. Initial plants are screened by several methods to ensure theintein modified cellulase DNA sequence has been transferred to thegenome and protein expression is active. Genetic screening is conductedby southern analysis on genomic DNA isolated from the transgenic plantusing the intein modified cellulase coding sequence as a probe, asdescribed by Ausubel, et. al. (1998), above. PCR is conducted usingprobes specific to the intein modified cellulase coding sequence and thetransgenic plant's genomic DNA as a template, as described above.Appearance of the appropriately sized band on an ethidium bromidestained gel verifies the presence of the intein modified cellulasecoding sequence. Direct sequencing of the plant's genomic DNA may alsobe performed. Protein production is monitored by western analysis usingantibodies specific to both the intein modified cellulase and the nativecellulase. In addition, enzymatic assays for cellulase activity areknown in the art and may be used to quantify the activity of theunspliced intein modified cellulase and the spliced cellulase.

Example 2 Production of Transgenic Douglase Fir

Expressing Intein-Modified Lignin Peroxidase

This example uses the same method for constructing the vector containingthe intein modified lignin peroxidase coding sequence as used in exampleone. The primary differences are in the A. tumefaciens plasmid employed,the native protein sequence that is modified, and the primers selectedto amplify the new intein modified lignin peroxidase coding sequence.

The lignin peroxidase gene (GenBank Accession #M37701) is amplified byPCR using genomic DNA from P. chrysosporium as a template. The primers5′-ATGGCCTTCAAGCAGCTCGTCGCAG-3′ [SEQ ID NO: 13]5′-TTAAGCACCCGGCGGCGGGGGGCTG-3′ [SEQ ID NO: 14]are are used in the PCR reaction as described in example one. Followingamplification, the resulting PCR product is analyzed using gelelectrophoresis on an agarose gel, along with molecular weight standardsas described in example one. After staining the gel with ethidiumbromide, the 1567 base pair (bp) band is cut from the gel with ascalpel, and purified from the gel as described above. After purifyingthe fragment from the gel, the fragment is analyzed using restrictiondigestion or sequencing for direct verification as described by Ausubel,et al., 1998.

After the gene is amplified, it is modified by insertion of the inteinsequence into the gene sequence. For this example, the same intein isused as in example one. The coding sequence of this intein is amplifiedin the same manner as described in example one. The resulting intein DNAsequence is purified by gel electrophoresis and analyzed as describedpreviously.

The two PCR fragments, one encoding the lignin peroxidase and oneencoding the intein polypeptide sequence, are joined. To ensure properintein splicing, the intein is inserted in frame next to a serineresidue of the lignin peroxidase such that this serine is on thecarboxylic acid side, of this intein's histidine-asparagine conservedresidues at the terminal 536 and 537 intein amino acid positions,respectively. The intein is inserted into the native protein, such thatthe serine residue of the native protein becomes the terminal C-exteinamino acid at the junction point between the native intein and C-exteinof the native protein. The intein is positioned within the nativeprotein such that its presence substantially reduces the activity of theresulting intein modified protein. In most circumstances virtually anyserine residue within or near the active site of the molecule will meetthis criterion, however some optimization may be necessary.

The intein modified protein sequence is produced using PCR the same asdescribed in example one, with the only difference being the choice ofprimers. The C-extein portion of the lignin peroxidase gene is amplifiedusing the cleaned gene product from the PCR reaction above, and thefollowing primers resulting in a 445 nucleotide sequence:5′-CTTTGGATTCCTCTATGCACATAATTCTCGCCCGCGACTCCCGCACCGCT-3′ [SEQ ID NO: 15]5′-TAAGCACCCGGCGGCGGGGGGCTGGAAGAGGAATATGTCAGCTGGGGGC-3′ [SEQ ID NO: 16]

The N-extein portion of the lignin peroxidase gene is amplified usingthe same template, by PCR using the following primers.5′-ATGGCCTTCAAGCAGCTCGTCGCAGCGATTTCCCTCGCACTCTCGCTCAC-3′ [SEQ ID NO: 17]5′-GAACCCATTCTTCCGGTAAAATGCTGTGTGGTCGGTCTGGATGCGGATCT-3′ [SEQ ID NO: 18]

The resulting sequence is 1196 nucleotides long. The intein codingsequence to be placed into the lignin peroxidase gene is amplified usingPCR as described in example one. In this reaction use a Pyrrococcus sppgenomic DNA template and the following primers:5′-AGATCCGCATCCAGACCGACCACACAGCATTTTACCGGAAGAATGGGTTC-3′ [SEQ ID NO: 19]5′-GCGGTGCGGGAGTCGCGGGCGAGAATTATGTGCATAGAGGAATCCAAAG-3′ [SEQ ID NO: 20]

The resulting sequence is 1661 nucleotides long. Once these reactionsare complete, the reaction products are electrophoresed on an agarosegel, purified from the gel, and analyzed as described above. The exteinand intein portions are joined as described in example one. In this casethe intein fragment is mixed with an equamolar portion of the C-exteinlignin peroxidase fragment. Combination of these fragments representsboth the template and primers required for the PCR reaction. PCR isperformed using the same reaction conditions as in example one. Oncecomplete, the PCR products are electrophoresed on a 1% agarose gel, andthe appropriate band, 2106 nucleotides long, is purified from the gel.The purified band is analyzed as described in example one. A smallamount of the purified reaction product is then quantified by measuringthe absorbance at 260 nm and 280 nm on a UV spectrophotometer.

The intein modified protein DNA coding sequence is completed with onemore PCR reaction. Equamolar amounts of the fused C-extein and inteinfragment just constructed are combined with the N-extein fragmentpurified previously. The PCR reaction is conducted using the sameconditions in the previous reactions. The reaction products areelectrophoresed on a 1% agarose gel, the appropriate band, 3302nucleotides long, is purified from the gel, and analyzed according tothe methods described in example one. The final intein modified proteincoding sequence has the complete intein sequence in frame, in the properorientation, within the lignin peroxidase coding sequence.

The intein modified lignin peroxidase coding sequence is cloned into aplant expression cassette. In this case, the pTiA6 plasmid is used withkanamycin resistance and lacking the octupine synthetase genes, butcontaining the octupine transcription control sequences. The inteinmodified lignin peroxidase is ligated into a restricted pTiA6 under theoctupine transcription control sequences (promoter and 3′polyadenylation site). A. tumefaciens K12X562 is transformed using theresulting ligated vector, and any suitable method known in the art(e.g., electroploration, or the calcium chloride method). Transformationmethods are described by Ausubel, et. al. (1998).

Douglas-fir is transformed, with the recombinant A. tumefaciens, andnodal segments or seeds sampled from these trees. The shootmultiplication and elongation is conducted as previously described(Gupta P K, Durzan, D J, “Shoot multiplication from mature trees ofDouglas-fir, and sugar pine,” Plant Cell Reports, 9:177-179, 1985) inculture on DCR basal medium plates. A culture of the recombinant A.tumefaciens is grown according to the method described in example one.For plant transformation, the regenerated shoots from culture,approximately 50 mm in length, or seeds are surface sterilized bytreatment with a 10% bleach and 0.1% Tween 20. Once sterilized, theshoots or seeds are aseptically rinsed with sterile, distilled, anddeionized water. The seeds or the shoots are transformed by firstwounding the epidermal tissue with a sterile needle or by cutting thesurface with a sterile scalpel. The wounded shoots or seeds are soakedin a culture of the recombinant A. tumefaciens at a cell concentrationof approximately 5×10⁸ cells per mL. After a 12 hour exposure to therecombinant A. tumefaciens culture, the shoots and seeds are cultured inDCR basal medium with 2.2% sucrose and 0.8% Bacto (Difco) agar. Theculture conditions include a 16 hour light cycle at 25° C., followed byand 8 hour dark cycle at 20° C. in a green house or growth chamber. Theregenerating plants are transferred to fresh plates every two to threeweeks. Once roots form, the explants are transferred to boxes containingsoil for four to five days prior to replanting in soil and full growthin a greenhouse or controlled plot of soil. The first year of growth isconducted within a green house under controlled temperature conditions,not exceeding 30° C.

The plants are screened using methods similar to those of example one,except specific to the lignin peroxidase protein or intein modifiedlignin peroxidase protein in the case of western analysis.

The resulting transgenic Douglas-fir species is grown indefinitely whileproducing the intein modified lignin peroxidase in high titer. Thelignin peroxidase is subsequently activated using the same methodsdescribed in example one because the same intein was employed formodification in this example.

Example 3 Fermentation Substrate Preparation Process

Using Plants Expressing Intein Modified Protein

In the case of example one, the transgenic poplar species can be used assubstrate for ethanol production via fermentation. For this process thetransgenic tree is harvested using a suitable tool, such as a chain sawor ax. The tree is subsequently pulped using a mechanical pulper. Thepulp is then placed it in a tank. After any necessary pretreatment hasbeen conducted, intein splicing is induced by raising the temperature ofthe tank and reducing the pH to a value of 4. Depending on thepretreatment used, intein splicing may be stimulated by the pretreatmentand thereby occur in parallel with that process operation. Once splicedthe native enzyme activity begins digesting the cellulose of the pulp,increasing the concentration of monosaccharides.

Following the induction of splicing, the contents of thesaccharification vessel are mixed in any proportion with native poplarpulp or other substrates, to facilitate cellulose degradation of thosesubstrates. The proportion of the mixing depends upon the cellulaseactivity of the transgenic poplar which is a function of the amount ofintein modified cellulase expressed in the plant, the efficiency ofsplicing, the efficiency of recombination, and the activity of therecombined, native cellulase on the substrate. Each one of thoseparameters has a broad spectrum of possible values and can be optimizedto facilitate process economics.

The resulting glucose is then filter sterilized from the degradedcellulose through a 0.22 (or less) μm filter, or heat sterilized inbatch or continuous mode through a heat exchanger. The sterilizedglucose is fed to a fermentation process, where it can be used as asubstrate for ethanol production as described in the literature. See, H.K. Sreenath and T. W. Jeffries, “Production of ethanol from woodhydrolysate by yeasts,” Bioresource Technology, 72(3): 253-260, 2000;Lisbeth Olsson and Barbel Hahn-Hagerdal, “Fermentation oflignocellulosic hydrolysates for ethanol production,” Enzyme andMicrobial Technology, 18(5):312-331, 1996; Kutluo O. Ulgen, et. al.,“Bioconversion of starch into ethanol by a recombinant Saccharomycescerevisiae strain YPG-AB”, Process Biochemistry, 37(10):1157-1168, 2002;M. Mete Altintas, et al, “Improvement of ethanol production from starchby recombinant yeast through manipulation of environmental factors,”Enzyme and Microbial Technology, 31(5):640-647, 2002; Farooq Latif, etal., “Production of ethanol and xylitol from corn cobs by yeasts,”Bioresource Technology, 77(1):57-63, 2001.

The fermentation process is conducted in batch, fed-batch, or continuousmodes.

Example 4 Plants Expressing an Intein Modified Protein used for AnimalFeed

A transgenic rapeseed is constructed following essentially the samemethods described in Examples 1 and 2 above, with the followingmodifications. In constructing the CIVPS or intein modified genesequence, the same intein coding sequence can be used, however in thiscase it is fused within the phytase expressed by Aspergillus ficuum. Inthis example the selected intein modified protein relies upon theacidity of the animal's stomach to induce protein splicing. The selectedphytase was chosen because of its high level of activity at low pH (vanOoijen et al. (2000), U.S. patent Publication No. 6,022,846). TheC-extein portion of the phytase is amplified using the following primersunder the same conditions as previously described.5′-CTTTGGATTCCTCTATGCACATAATTTCCTTCGACACCATCTCCACCAGCA-3′ [SEQ ID NO:21] 5′-CTAAGCAAAACACTCCGCCCAATCACCGCCAGATCTGGCAAAGCTCAACC-3′ [SEQ ID NO:22]

The resulting sequence is 627 nucleotides long. The intein sequence isamplified using primers under the same conditions as previouslydescribed. 5′-AGTGACCTACCTCATGGACATGTGCAGCATTTTACCGGAAGAATGGGTTC-3′ [SEQID NO: 23] 5′-GCTGGTGGAGATGGTGTCGAAGGAATTATGTGCATAGAGGAATCCAAAG-3′ [SEQID NO: 24]

Finally the N-extein is amplified using primers resulting in a PCRfragment 928 nucleotides long.5′-ATGGGTGTCTCTGCCGTTCTACTTCCTTTGTACCTCCTGTCCGGAGTATG-3′ [SEQ ID NO: 25]5′-GAACCCATTCTTCCGGTAAAATGCTGCACATGTCCATGAGGTAGGTCACT-3′ [SEQ ID NO: 26]

The resulting DNA fragments are cleaned and analyzed, then combinedusing PCR and the associated methods described in Examples 1 and 2. Thisprocedure results in the intein modified phytase coding sequence. Thefinal composite intein modified phytase sequence is then amplified,cleaned and analyzed as described in Examples 1 and 2. The inteinmodified phytase DNA coding sequence is cloned into the same expressioncassette, and used to transform A. tumefaciens as described in Example2. Rapeseed stem segments are transformed using the resultingrecombinant A. tumefaciens. Transformation occurs substantially asdescribed in Examples 1 and 2 with the following modifications. Therapeseed stem segments are surface sterilized from five to six week-oldplants using a 20% bleach solution for 25 minutes at room temperature.Following sterilization, the stem segments are aseptically rinsed withsterile, distilled, and deionized water. The segments are preconditionedby incubation for 24 hours on Murashige-Skoog medium supplemented with 1mg/L of BAP. Once the 24 hours has transpired, the stem segments areincubated for 48 hours with the newly transformed strain of A.tumefaciens containing the intein modified phytase. Following thisincubation step, regenerate transgenic plants and select them using thekanamycin resistance marker, following substantially the same proceduresdescribed in Examples 1 and 2. Confirmation of incorporation of theintein modified phytase can also be conducted as described in Examples 1and 2.

The resulting transgenic rapeseed is grown in an approved area accordingto local legislation. The rapeseed is harvested when it's mature andused to supplement animal feed. Conversely, the rapeseed can be grown ongrazing land for the animals since intein splicing should occurspontaneously in the animal's stomach, allowing for activation of thephytase activity.

Example 5 Production of Transgenic Maize Expressing an Intein Modified

Cellulase and Utilization in the Production of Ethanol

This example illustrates one way in which the invention may bepracticed. Here, transgenic corn is constructed and used as a substratefor ethanol processing, or as a substrate in other fermentations. Inthis example the intein modified gene sequence of Example 1 is againused for demonstration. The growth of Zea mays friable, embryogenic typeII callus cultures is initiated from immature embryos, approximately 1.6mm to 1.8 mm in length, from greenhouse grown A188 (University ofMinnesota, Crop Improvement Association)×B73 (Iowa State University)plants. After harvest, fragments are surface sterilized using 50%bleach, for 25 minutes at room temperature, and then washed withsterile, distilled, deionized water. New cultures are aspeticallyinitiated from the harvested fragments and maintained under no more than10 μE m⁻² s-⁻¹ light, at 24° C., on modified N6 medium (Chu, et al.,(1975), “Establishment of an Efficient Medium for Anther Culture of Ricethrough Comparative Experiments on Nitrogen Sources,” Sci. Sin.,18:659-668) at pH 5.8, with 2 mg/L glycine, 2.9 g/L L-proline, 1 mg/L2,4-dichlorophenoxyacetic acid (2,4-D), 100 mg/L casein hydrolysate, 20g/L sucrose, and solidified with 2 g/L Gelgro (ICN Biochemicals).

After approximately two weeks of incubation, the cultures are manuallyevaluated for proper morphology. This entails visual observation forfriable consistency in the presence of well-characterized somaticembryos. Proliferations demonstrating proper morphology are transferredto fresh modified N6 medium (described above). Tissues resulting withthe proper morphology are routinely subcultured every two to threeweeks, until the microprojectile bombardment is prepared. The desiredintein modified gene sequence and expression vector can be constructedas described in Example 1. In this example, the preferred expressionvector also has the following alterations. Replace the kanamycinresistance marker with a hygromycin resistance marker using methodsknown in the art (for example, PCR of the hygromycin resistance markerfrom a suitable template, DNA endonuclease restriction of the vector,followed by purification, and ligation of the hygromycin resistancemarker) as described by Ausubel, et. al., 1998. Once constructed, thevector is precipitated in a 1:1 molar ratio onto either tungsten(average diameter 1.2 μm, GTE Sylvania), or gold, particles. As withother steps in this procedure, the precipitation parameters may requiresome minor optimization. The precipitation is performed by combining1.25 mg of the tungsten particles, and 25 μg of the vector DNA insolution with 1.1 M CaCl₂ and 8.7 mM spermidine at a total volume of 575μL. The precipitate is vortexed for 10 minutes at 0° C. Once vortexed,the mixture is centrifuged at 500×g for five minutes. Aftercentrifugation, the supernatant, approximately 550 μL, is removed andthe remaining 25 μL of precipitate is dispensed in 1 μL aliquots ontomacroprojectiles (Biolistics, Inc, Ithaca, N.Y.) for bombardment asdescribed by Klein et al. (1987), except for the changes noted above.All manipulations are performed aseptically and on ice.

Once the biolistic projectiles are ready, the desired plant tissues areprepared for the bombardment procedure. Any number of callus clumps areaseptically arranged, each weighing 50 mg (wet weight), in an x-patternnear the center of a sterile 60×15 mm petri dish (Falcon 1007). Severaldishes should be prepared for each bombardment procedure. These dishesare each paced in turn, 5 cm below the stopping plate of themicroprojectile instrument. The dishes are centered below the device,with the lids removed, and a 3×3 mm mesh screen covering the top of theplate. The mesh screen helps contain bombarded tissue within the dishduring the procedure. The tissue bombardment is performed with themicroprojectile instrument as described by the manufacturer'sinstructions; commercial microprojectile instruments are availablethrough Bio-Rad (Hercules, Calif.). Following bombardment, the callusare transferred to fresh modified N6 medium plates and cultured underthe same conditions used above.

The selection of transformed cells for subsequent regeneration is beganafter two days of culture. The callus plates subjected to thebombardment procedure are aseptically transferred to fresh, sterile,modified N6 medium plates formulated to a final concentration of 10 mg/Lhygromycin B (Calbiochem). After two weeks of exposure, all callus areaseptically transferred from the selective plates to fresh, sterile,modified N6 medium plates formulated to a final concentration of 50 mg/Lhygromycin B. This transfer is conducted so that only five 30 mg piecesof callus are contained on a single plate, resulting in an expansion ofthe number of plates used. Following three weeks on the 50 mg/Lhygromycin B plates, all callus are aseptically transferred to fresh,sterile, modified N6 medium plates formulated to a final concentrationof 60 mg/L hygromycin B. After two weeks of incubation, the callus areinspected for proliferating clumps. Selected proliferating clumps aretransferred to a modified Murashige-Skoog medium supplemented with 0.5mg/L thiamine-HCl, 0.75 mg/L 2,4-D, 50 g/L sucrose, 150 mg/Lasparagines, and 2.0 g/L Gelgro.

At this point it is prudent to ensure transformation of the selectedplants. The presence of the intein modified cellulase is verified usingthe methods described in Examples 1 and 2. In this case, either or bothof the intein modified cellulase coding sequence, and the hygromycinresistance marker can be used as the subject of the transformationvalidation, using methods known in the art, as described by Ausubel, etal., 1998. After two weeks on the modified Murashige-Skoog medium, theplates are exposed to a light cycle incubation regimen composed of 14hours of light, followed by 10 hours of dark, at 24° C. Plantlets thatform are aseptically transferred to 1 L, wide mouthed Erlenmeyer flaskscontaining 100 mL of the modified Murashige-Skoog medium. The resultingplants are transferred to vermiculite for one to two weeks prior toplantation in soil and growth to maturity. The mature plants areanalyzed substantially as described in Example 1 to ensure stabletransformation of the intein modified protein sequence, andpreferentially, expression of the intein modified cellulase.

The resulting mature plants may be cross-pollinated using standardtechniques. This can be done either between transformed plants, orbetween a single transformed plant and an untransformed plant. Theprogeny resulting from the breeding are screened for containment of theintein modified cellulase, as well as the hygromycin resistance marker.Note, at this point the hygromycin resistance marker used in theselection is no longer an essential element for the use and applicationof the constructed transgenic corn plants. So long as the inteinmodified cellulase sequence is contained, retention of the hygromycinresistance marker is not an essential component of the transgenic corn.Seed can be harvested from the fertile transgenic plants and used forplant expansion. The resulting transgenic plants can be grown for use inprocesses similar to those described in Example 3. The process using atransgenic corn species expressing multiple intein modified proteins,would have the economic advantages of utilizing both the starch andcellulosic portions of the corn plant, consolidating the pretreatment,saccharification, and fermentation steps, and decreased energy and rawmaterial input costs. Effective use of this process for the productionof ethanol would be enabled by the inclusion of the intein modifiedproteins in the transgenic plant.

The enclosed examples do not in any way limit the scope of this patent,as they solely provided to help illustrate applications of the inventiondisclosed in this patent. Other variations are possible as an artisanwould know, and are included within the four corners of this invention.

Bibliography

Dale, Biotechnol. Prog. 15:775-776 (1999).

Committee on Biobased Industrial Products, Biobased Industrial Products:Priorities for Research and Commercialization, National Academy Press,Washington D.C. (1999).

Cameron, et al., Biotechnol. Prog. 14:116-125 (1998).

Park, et al., Biotechnol. Prog. 14:699-704 (1998).

Taylor, et al., Biotechnol. Prog. 16:541-547 (2000).

Poirier, Nature Biotechnology 17:960-961 (1999).

Lynd, Biotechnol. Prog. 15:777-793 (1999).

Ingram, Biotechnol. Prog. 15:855-866 (1999).

Aspergren, et al., Molecular Breeding 1:91-99 (1995).

Mansfield, et al., Biotechnol. Prog. 15:804-816 (1999).

Evans, et al., Protein Science 7:2256-2264 (1998).

Perler, et al., Nucl. Acids Res. 22:1125-1127 (1994).

Xu, et al., EMBO 15:5146-5153 (1996).

Wood, et al., Biotechnol. Prog. 16:1055-1063 (2000).

Clarke, P.N.A.S. (USA) 91:11084-11088 (1994).

Derbyshire, et al., P.N.A.S. (USA) 95:1356-1357 (1998).

Perler, et al., Nucleic Acids Res. 22:1125-1127 (1994).

Wallace, C. J., Protein Sci. 2:697-705, (1993).

Xu, et al., Cell 75:1371-1377 (1993).

Pietrokovski, S., Protein Sci. 3735:2340-2350 (1994).

Ausubel, et al., “Current Protocols in Molecular Biology,” Wiley, N.Y.(1998).

Draper, et al., “Plant Genetic Transformation and Gene Expression: ALaboratory Manual,” Blackwell Scientific Publications, Boston (1998).

Potrykus, et al., “Gene Transfer to Plants,” Springer, N.Y. (1995).

Guilley, et al., Cell, 30:763 (1982).

Higgins, T. J. V., Annu. Rev. Plant Physiol. 35:191(1984).

Coruzzi, et al., EMBO J. 3:1671 (1984).

Tingey, et al., EMBO J. 6:3565 (1987).

Ryan, et al., Nuc. Acids Res. 17:3584 (1989).

Rocha-Sosa, et al., EMBO J. 8:23 (1989).

Wenzler, et al., Plant Mol. Biol. 12:41 (1989).

Bird et al., Plant Mol. Biol. 11:651 (1988).

Brederode, et al., Nucl. Acids Res. 8:2213 (1980).

Smeekens, et al., T.I.B.S. 15:73 (1990).

van den Broeck et al., Nature 313:358 (1985).

Schreier, et al., EMBO J. 4:25 (1985).

Tague, et al., Plant Phys. 86:506 (1988).

Von Heijne, G., J. Mol. Biol. 189:239 (1986).

Sijmons, et al., Bio/Technol. 8:217 (1990).

Gordon-Kamm, et al., The Plant Cell 2:603 (1990).

Shimamoto, et al., Nature 338:274 (1989).

Horsch, et al., Science 2:1229 (1985).

Ziegler, et al., Molecular Breeding 6:37-46 (2000).

Dai, et al., (a), Molecular Breeding 6:277-285 (2000).

Dai, et al., (b), Molecular Breeding 9:43-54 (2000).

Montvalvo-Rodriguez, et al., Biotech. and Bioeng. 2:151-159 (2000).

U.S. Pat. No. 6,022,846.

Chu, et al., Sci. Sin. 18:659-668 (1975).

Klein, et al., Nature 327:70-73 (1987).

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

1. A recombinant plant, or plant part, plantlet, tissue, cell,sub-cellular fraction, seed, seedling, protoplast, progeny ordescendent, comprising an expression construct(s) that encode(s) atleast one modified protein comprising a target protein(s) or proteinsegment(s), which is(are) fused, either internally or terminally, to acontrollable intervening protein sequence(s) (CIVPS) or inteinsequence(s) or segment(s) thereof, or to an amino terminus(i) or acarboxyl terminus(i) thereof.
 2. The plant, or plant part, plantlet,tissue, cell, sub-cellular fraction, seed, seedling, protoplast, progenyor descendent of claim 1, wherein each expression construct comprises,operatively linked to one another, a first nucleic acid segment(s)encoding a target protein(s), and a second nucleic acid segment(s)encoding a CIVPS or intein sequence(s), and optionally a selectablemarker(s) or reporter gene(s) and/or a promoter(s).
 3. The plant, orplant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent of claim 1, wherein themodified protein(s) is(are) expressed constitutively.
 4. The plant, orplant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent of claim 1, whichinductively expresses at least one modified protein(s); and optionallywherein a stimulus(i) triggers splicing of the modified protein.
 5. Theplant, or plant part, plantlet, tissue, cell, sub-cellular fraction,seed, seedling, protoplast, progeny or descendent of claim 4, whereinthe stimulus(i) comprise(s) a change in pH, osmolality, temperature, afertilizer(s), pesticide(s), chemical(s), light, and/or sound.
 6. Theplant, or plant part, plantlet, tissue, cell, sub-cellular fraction,seed, seedling, protoplast, progeny or descendent of claim 1, wherein atleast one modified protein(s) is(are) expressed at a pre-determinedpoint of the plant life cycle; in at least one specific tissue or partthereof; and/or in at least one specific sub-cellular compartment(s);and/or expressed and secreted extracellularly.
 7. The plant, or plantpart, plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent of claim 2, wherein the at least oneselectable marker confers resistance to a chemical comprisingbromoxynil, 2,2-dichloropropionic acid, G418, glyphosphate, haloxyfop,hygromycin, imidazoline, kanamycin, methotrexate, neomycin,phosphinothricin, sethoxydim, 2,2-dichloropropionic acid, glyphosphate,hygromycin, trichothecne, sulfonylurea, s-triazine and/ortriazolopyrimidine; and/or the promoter precedes the at least one CIVPSor intein-modified protein polynucleotide.
 8. The plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent of claim 7, which is tolerant orresistant to normally toxic levels of at least one chemical(s).
 9. Theplant, or plant part, plantlet, tissue, cell, subcellular fraction,seed, seedling, protoplast, progeny or descendent of claim 1, which isfertile, and has at least one heritable CIVPS or Intein-modified proteinencoding polynucleotide sequence(s).
 10. The plant, or plant part,plantlet, tissue, cell, subcellular fraction, seed, seedling,protoplast, progeny or descendent of claim 1, wherein the plant is notfertile.
 11. An inbred genetically recombinant plant, or plant part,plantlet, tissue, cell, subcellular fraction, seed, seedling,protoplast, progeny or descendent of claim
 1. 12. A hybrid geneticallyrecombinant plant, or plant part, plantlet, tissue, cell, subcellularfraction, seed, seedling, protoplast, progeny or descendent of claim 1.13. The plant, or plant part, plantlet, tissue, cell, subcellularfraction, seed, seedling, protoplast, progeny or descendent of claim 4,wherein the spliced protein(s) changes(s) the content or activity of atleast one pre-determined plant component(s).
 14. The plant, or plantpart, plantlet, tissue, cell, subcellular fraction, seed, seedling,protoplast, progeny or descendent of claim 13, wherein the plantcomponent(s), whose content(s) change(s) comprise(s) cellulose,hemicellulose, lignin, proteins, ribonucleic acids, starch, pectinand/or lipids.
 15. The plant, or plant part, plantlet, tissue, cell,subcellular fraction, seed, seedling, protoplast, progeny or descendentof claim 13, wherein the plant component(s), whose activity(ies)change(s) comprise(s) protein, polypeptide, enzyme, hormone, receptor.16. The plant, or plant part, plantlet, tissue, cell, subcellularfraction, seed, seedling, protoplast, progeny or descendent of claim 1,wherein the CIVPS or intein sequence(s) and the target protein(s) orprotein segment(s) form at least one splice junction(s).
 17. The plant,or plant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent of claim 1, wherein theamino acid residue at the carboxyl terminus of the splice junction(s)comprise(s) an amino acid residue provided with a hydroxyl or asulfhydryl side chain(s).
 18. The plant, or plant part, plantlet,tissue, cell, sub-cellular fraction, seed, seedling, protoplast, progenyor descendent of claim 16, wherein the splice junction(s) downstream ofthe CIVPS or intein sequence(s) or segment(s) thereof comprises an aminoacid residue lacking a hydroxyl or sulfhydryl side chain(s) at the aminoterminus of the target protein or protein segment(s).
 19. The plant, orplant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent of claim 16, wherein thesplice junction(s) upstream of the CIVPS or intein sequence(s) orsegment(s) thereof comprises an amino acid residue having a hydroxyl ora sulfhydryl side chain at the amino terminus of the CIVPS or inteinsequence(s) or protein segment(s).
 20. The plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent of claim 16, wherein the splicejunction(s) upstream of the CIVPS or intein sequence(s) or segment(s)thereof comprise(s) a cysteine.
 21. The plant, or plant part, plantlet,tissue, cell, sub-cellular fraction, seed, seedling, protoplast, progenyor descendent of claim 16, wherein the splice junction(s) downstream ofthe CIVPS or intein sequence(s) or segment(s) thereof comprise(s)His-Asn at the carboxyl terminus(i) of the CIVPS or intein sequence(s)or segment(s) thereof, and an amino acid residue having a hydroxyl orsulfhydryl side chain at the amino terminus(i) of the adjoiningregion(s) of the target protein(s).
 22. The plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent of claim 16, wherein the splicejunction(s) downstream of the CIVPS or intein sequence(s) or proteinsegment(s) thereof comprise(s) Asp at the carboxyl terminus(i) of theCIVPS or intein sequence(s) or segment(s) thereof, and an amino acidresidue having a hydroxyl or sulfhydryl side chain at the aminoterminus(i) of the adjoining region(s) of the target protein(s) orprotein segment(s).
 23. The plant, or plant part, plantlet, tissue,cell, sub-cellular fraction, seed, seedling, protoplast, progeny ordescendent of claim 22, wherein the Asp at the carboxyl terminus(i) Aspis replaced by an amino acid lacking a carboxyl or an amino sidechain(s).
 24. The plant, or plant part, plantlet, tissue, cell,subcellular fraction, seed, seedling, protoplast, progeny or descendentof claim 1, wherein the CIVPS or intein sequence(s) or segment(s)thereof comprises an externally controllable Saccharomyces CIVPS orintein sequence(s).
 25. The plant, or plant part, plantlet, tissue,cell, sub-cellular fraction, seed, seedling, protoplast, progeny ordescendent of claim 1, wherein the CIVPS or intein sequence(s) is(are)inserted immediately before Ser, Thr or Cys of the target protein(s) orprotein segment(s).
 26. The plant, or plant part, plantlet, tissue,cell, sub-cellular fraction, seed, seedling, protoplast, progeny ordescendent of claim 1, wherein the CIVPS or intein amino or carboxyterminus(s) comprise(s) Ser, Thr or Cys.
 27. The plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent of claim 1, wherein the protein isexpressed by a microorganism.
 28. A method for producing the recombinantplant, or plant part, plantlet, tissue, cell, sub-cellular fraction,seed, seedling, protoplast, progeny or descendent having an expressionconstruct(s) that encode(s) at least one modified protein that comprisesa target protein(s) or protein segment(s), which is(are) fused, eitherinternally or terminally, to a controllable intervening proteinsequence(s) (CIVPS) or intein sequence(s) or segment(s) thereof, or toan amino terminus(i) or a carboxyl terminus(i) thereof, the methodcomprising providing an expression construct that encode(s) at least onemodified protein comprising a target protein, or protein segment(s),which is(are) fused, either internally or terminally, to a CIVPS orintein sequence(s) or segment(s) thereof, or to an amino terminus(i) ora carboxyl terminus(i) thereof; transforming a plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent, with the expression construct; andregenerating a genetically recombinant plant, or plant part, plantlet,tissue, cell, sub-cellular fraction, seed, seedling, protoplast, progenyor descendent, from the transformed plant, or plant part, plantlet,tissue, cell, sub-cellular fraction, seed, seedling, protoplast, progenyor descendent, that encode(s) at least one modified protein sequence(s).29. The method of claim 28, wherein the transformation comprises astable transformation.
 30. The method of claim 28, wherein theregeneration step is conducted by at least one of breeding of arecombinant plant, or plant part, plantlet, tissue, cell, sub-cellularfraction, seed, seedling protoplast, progeny or descendent; crossing ofa recombinant plant, or plant part, plantlet, tissue, cell, sub-cellularfraction, seed, seedling, protoplast, progeny or descendent and anon-genetically recombinant plant, or plant part, plantlet, tissue,cell, sub-cellular fraction, seed, seedling, protoplast, progeny ordescendent; or back-crossing of two genetically recombinant plant, orplant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent.
 31. The method of claim 28,wherein the expression construct comprises at least one of promoter,selectable marker, resistance marker, heritable marker, poly-adenylationsequence, repressor, enhancer, localization sequence, or signalingsequence.
 32. The method of claim 28, wherein the plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent is transformed with the expressionconstruct by at least one of viral transformation, bombardment withDNA-coated microprojectiles, liposomal gene transformation, bacterialgene transfer, electroporation, or chemical gene transformation.
 33. Themethod of claim 28, wherein the transformed plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent is selected with the aid of at leastone of a selectable marker, or resistance marker.
 34. The method ofclaim 28, wherein the genetically recombinant plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent is regenerated from one of transformedembryogenic tissue(s); plant protoplasts; cells derived from immatureembryos; or transformed seeds.
 35. A method for producing seedexpressing a modified protein(s), comprising obtaining the geneticallyrecombinant plant, or plant part, plantlet, tissue, cell, subcellularfraction, seed, seedling, protoplast, progeny or descendent comprisingthe method of claim 28; culturing or cultivating the geneticallyrecombinant plant, or plant part, plantlet, tissue, cell, subcellularfraction, seed, seedling, protoplast, progeny or descendent; andobtaining therefrom seed that expresses a CIVPS or Intein-modifiedprotein(s).
 36. A method for producing a protein(s) or proteinsegment(s) from a recombinant transformed plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent expressing the protein(s) or proteinsegment(s), comprising conducting the method of claim 28; harvesting themodified protein(s) or protein segment(s) from the transformed plant, orplant part, plantlet, tissue, cell, sub-cellular fraction, seed,seedling, protoplast, progeny or descendent; and optionally purifyingthe protein.
 37. A method of producing a target protein(s) or proteinsegment(s), comprising producing a first modified protein(s) or proteinsegment(s), wherein the amino terminus of a CIVPS or intein sequence(s)or segment(s) thereof is(are) fused to the carboxyl terminus(i) of atarget protein(s) or protein segment(s) by the method of claim 36;producing a second modified protein(s) comprising a segment(s) of theCIVPS or intein sequence(s); and contacting first and second modifiedproteins under conditions effective for trans cleavage of the CIVPS orintein sequence(s) or segment(s) thereof by the second modifiedprotein(s).
 38. A method of producing a target protein(s), comprisingproducing a first modified protein(s), wherein the carboxyl terminus ofa CIVPS or intein sequence(s) or protein segment(s) thereof is(are)fused to the amino terminus(i) of the target protein(s) or proteinsegment(s) by the method of claim 36; similarly producing a secondmodified protein(s) or protein segment(s) comprising a segment(s) of theCIVPS or intein sequence(s); and contacting first and second modifiedproteins under conditions effective for trans cleaving the CIVPS orintein sequence(s) or segment(s) thereof from the first modifiedprotein(s) or protein segment(s).
 39. The method of claim 38, whereinthe cleavage is induced by a change in at least one of temperature,light, or pH, addition/removal of chemical that facilitates/inhibitssplicing or blocking of cleavage, amino acid dephosphorylation ordeglycosylation, or contact/removal of peptide or peptidomimetic thatactivates/blocks splicing/cleavage.
 40. A method for producing amodified protein comprising a target protein(s) or protein segment(s)fused, either internally or terminally, to a CIVPS or intein sequence(s)or segment(s) thereof, or to its amino terminus(i) or carboxylterminus(i), comprising obtaining an expression construct encoding atarget protein having an in-frame fused CIVPS or intein sequence(s) orsegment(s) thereof, or its amino terminus(i) or carboxyl terminus(i);transforming a host plant cell(s) with the expression construct; andculturing the transformed plant host cell under conditions effective forexpressing the modified protein.
 41. The method of claim 40, wherein inthe expression construct, the at least one first nucleic acid segment(s)encoding the CIVPS or intein sequence(s) or segment(s) thereof is(are)fused to the 5′-end or 3′-end of the at least one second nucleic acidsegment(s) encoding the target protein(s) or protein segment(s).
 42. Themethod of claim 40, wherein the CIVPS or intein sequence(s) orsegment(s) thereof is(are) capable of effecting, either in cis or intrans, at least one of excision, cleavage, ligation, excision-ligation,cleavage-ligation, or cyclization.
 43. The method of claim 40, whereinprotein splicing is induced by at least one of change of temperature,light or pH, addition/removal of chemical reagent thatfacilitates/inhibits splicing or cleavage, amino acid dephosphorylationor deglycosylation, or in vitro or in vivo contact with/removal ofpeptide or peptidomimetic activating/blocking splicing or cleavage. 44.The method of claim 40, wherein the amino or carboxy terminus(i) of theCIVPS or intein sequence(s) or segment(s) thereof comprise(s) Ser, Thror Cys.
 45. The method of claim 40, wherein the carboxyl terminus of theCIVPS or intein sequence(s) or segment(s) thereof comprise(s) Asppreceding Ser, Thr or Cys of the target protein(s) or proteinsegment(s).
 46. The method of claim 40, wherein the expression constructfurther comprises at least one of promoter, selectable marker,resistance marker, heritable marker, poly-adenylation sequence,repressor, enhancer, localization sequence, or signaling sequence; andoptionally further comprising transforming the plant, or plant part,plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent with the expression construct by atleast one of viral transformation, bombardment with DNA-coatedmicroprojectiles, liposomal gene transfer, bacterial gene transfer,electroploration, or chemical gene transformation.
 47. The method ofclaim 40 wherein: the transformed plant, or plant part, plantlet,tissue, cell, sub-cellular fraction, seed, seedling, protoplast, progenyor descendent is(are) selected through their expression of the modifiedprotein gene sequence; and the genetically recombinant plant, or plantpart, plantlet, tissue, cell, sub-cellular fraction, seed, seedling,protoplast, progeny or descendent is(are) regenerated from transformedembryogenic tissue, plant protoplasts, cells derived from immatureembryos or transformed seeds.